Bisphosphonates for modified nucleotide synthesis and related chemistry

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Content BISPHOSPHONATES FOR MODIFIED NUCLEOTIDE SYNTHESIS AND RELATED CHEMISTRY by Rehana Ismail A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (CHEMISTRY) August 2009 Copyright 2009 Rehana Ismail ii DEDICATED To My Family iii Acknowledgements This dissertation would have been impossible without the generous contribution of my mentor, Dr. G. K. Surya Prakash. He has been a constant guide in my time at the Loker Hydrocarbon Institutes. I would like to thank him for giving me the opportunity to work with him and also for his patience and kindness. I would also like to thank Prof. George A. Olah for his endless knowledge and wisdom in chemistry as well as in life. The group meetings were especially inspiring and enjoyable due to his presence. I would like to acknowledge my committee members, Dr. William Weber, Dr. Kathrine Shing, Dr. Golam Rasul and the late Dr. Robert Bau for their advice. Over the years I have worked with various people in my research projects. Part of this thesis was carried out with collaboration with other members of the group and also with other groups in the department of Chemistry. Here I would like to acknowledge Dr. Markus Etzkorn, whom I worked in the beginning on a carbocation project; Dr. Thomas Mathew and Dr. Chiradeep Panja, whom I collaborated in the α‐aminophosponate and halogenation projects; Dr. Roman Kultyshev, Dr. Petr Beir and Micheal Zibinsky in the modified nucleotide project. I would also like to acknowledge the people from the McKenna & Bau Groups for helping us with the nucleotide project. I would like to take this opportunity to thank the faculty members at California State University, Los Angeles, Dr. Mathias Selke, Dr. Donald Paulson and iv Dr. Carlos Gutierrez, for inspiring me to come for graduate studies. I would also like to thank Vicki Kubo Anderson at California State University, Los Angeles for her constant encouragement since the beginning of my time here at USC. Through my career in Loker Hydrocarbon Research Institute I have acquired realm of knowledge from my peers and friends in the institute. Their presences have enriched my learning and development as a scientist and as a person. Here I would like to especially acknowledge Dr Habiba Vaghoo, Dr. Gabriale Foggasy, Dr. Alain, Goeppert, Dr. Akhisa Saitoh, Dr. Robert Aniszfeld, Dr. Kiah Smith, Dr. Patrice Batamack, Dr. Csaba Weber, Dr. Juan Carlos Colmenares, Ms. Gloria Canada, Dr. Matthew Moran, Dr. Farzaneh Paknia, Kevin, Dr Sujith Chacko, Clement, Fang, Jessy May, Carol, Dr. Chaunfa Ni, Charlie, Arjun Narayan Anthon,Dr. Fabrizio Pertusati, Dr. Somesh Kumar, Dr. Parag Jog ,Dr. Miklos Czuan. I would like to thank my friends outside of the Loker Hyrodrocarbon Institute for their encouragements. I would like to especially acknowledge Stephen DeSalvo, Dr Melania Cosmina Oana, Daisy Khuu, Dr. Jose Nunez, Hala Mohammed, Angeliki Metallinou, Sarah Ahmed and Dorte Pederson. I would like to thank all my sisters, Nasrin Hoque (Boro Apa), Nazneen Yousuf (Nazu Apa), Nurjahan (Zharna Apa), Marjahan (Munni Apa), Yasmin and my brother Mizan for their love and support. I would also like to acknowledge my brother‐in‐laws, Emdadul Hoque, Sheikh Yousuf and Dipu who have treated me like their little sister. I would like to especially thank my brother‐in‐law, Sheikh Yousuf and Nazu Apa for taking me into their home and giving me a loving and supporting v environment, when I came to the United State. Their daughter Sara and Farah made my life less stressful. Last but not the least; I would like to thank my parents for being constant supporter in my life. My father has been the foundation of our family his life has inspired many of us to try to reach for our full potentials. vi Table of Contents Dedication ..................................................................................................................................................... ii Acknowledgements .................................................................................................................................. iii List of Tables ............................................................................................................................................ viii List of Figures ............................................................................................................................................. ix List of Schemes .......................................................................................................................................... xi Abstract ...................................................................................................................................................... xiii Chapter 1. Introduction to Phosphorus Chemistry ..................................................................... 1 1.1 Characteristics of Phosphorus Compounds ....................................................................... 1 1.2 Phosphorus in Synthesis ............................................................................................................. 9 1.3 Phosphorus Compounds in Biological System ............................................................... 13 1.4 Phosphorous in Nerve Agents, Insecticides and Herbicides .................................... 17 1.5 Organophosphorus Compound in Medicine ................................................................... 20 1.6 Phosphorus in Materials. ......................................................................................................... 23 1.7 Conclusions .................................................................................................................................... 24 1.8 Chapter 1 References ................................................................................................................ 25 Chapter 2. Synthesis of Fluorinated α‐Aminophosphonates using Gallium Triflate as catalyst. .................................................................................................................................................. 30 2.1 Introduction .................................................................................................................................. 30 2.2 Results and Discussions ........................................................................................................... 33 2. 3 Conclusions................................................................................................................................... 40 2.4.1 General ........................................................................................................................................ 41 2.4.2 Typical synthesis of α‐Aminophosphonates .............................................................. 42 2.5 Spectral Data: ................................................................................................................................ 42 2.6 Crystal Structure Data ............................................................................................................... 45 2.7 Chapter 2 NMR Spectra ............................................................................................................ 52 2. 8 Chapter 2 References ............................................................................................................... 66 Chapter 3. Modified Nucleotide Triphosphate Analogs ......................................................... 69 3.1 Introduction .................................................................................................................................. 69 3.2 Result and Discussion ............................................................................................................... 72 3.2.1 Bisphophonic Acid Analogs ............................................................................................... 75 3.2.2 Triphosphoric Acid Analogs .............................................................................................. 80 3.2.3 Monophonic Acid Analog of Bisphophonic Acid ....................................................... 82 vii 3.2.4 Modified Nucleotide Analog .............................................................................................. 84 3.3 Conclusion ...................................................................................................................................... 86 3.4 Experimental ................................................................................................................................. 86 3.4.1 General ........................................................................................................................................ 86 3.4.2 Synthesis Relevance to Chapter 3 ................................................................................... 87 3.5 Chapter 3 NMR and HPLC Spectra ....................................................................................... 97 3.6 Chapter 3 References .............................................................................................................. 108 Chapter 4. Halogenated Trimethylsilane and Nitrate Salt as an Efficient Reagent System for the Direct α‐Halogenation of Carbonyl Compounds ...................................... 111 4.1 Introduction ................................................................................................................................ 111 4.2 Results and Discussion ........................................................................................................... 112 4. 3 Conclusions................................................................................................................................. 120 4. 4 Experimental ............................................................................................................................. 121 4.4.1 General ...................................................................................................................................... 121 4.4.2 General Procedure for the Halogenation Reaction ................................................ 121 4.5 Chapter 4 References .............................................................................................................. 122 Bibliography ............................................................................................................................................ 125 viii List of Tables Table 1.1 31 P NMR Shifts of Various Organophosphorus Compounds. .............................. 6 Table 1.2 The Biochemical Role of Phosphorus‐Containing Compounds ...................... 14 Table 2.1 Results of the Kabachnik‐Fields Reaction for Fluorinated α‐ Aminophosphonates .............................................................................................................................. 36 Table 2.2 Result for Imine Formation Under Conventional Heating ................................ 39 Table 2.3 Result for Imine Formation Under Microwave Conditions .............................. 40 Table 2.4 Crystal data and structure refinement for C 13 H 17 FN 2 O 5 P ................................. 45 Table 2.5 Atomic Coordinates ( x 10 4 ) and Equivalent Isotropic Displacement Parameters (Å 2 x 10 3 )for C 13 H 17 FN 2 O 5 P. U(eq) is Defined as One Third of the Trace of the Orthogonalized U ij Tensor ...................................................................................................... 47 Table 2.6 Bond lengths [Å] and angles [°] for C 13 H 17 FN 2 O 5 P ............................................... 48 Table 2.7 Anisotropic displacement Parameters (Å 2 x 10 3 ) for C 13 H 17 FN 2 O 5 P. The Anisotropicdisplacement Factor Exponent Takes the Form: ‐2π 2 [ h2 a*2U11 + ... + 2 h .............................................................................................................................................................. 50 Table 2.8 Hydrogen coordinates ( x 10 4 ) and Isotropic displacement parameters (Å 2 x 10 3 ) for C 13 H 17 FN 2 O 5 P. .............................................................................................................. 51 Table 4.1 α‐Chlorination of Acetophenones with TMSCl‐Nitrate System .................... 116 Table 4.2 α‐Bromination of Acetophenones With TMSBr‐Nitrate System .................. 118 ix List of Figures Figure 1.1 Six‐ and Five‐Coordinate Phosphorus Compounds. ............................................. 3 Figure 1.2 Various Phosphoralkenes. ............................................................................................... 5 Figure 1.3 Chiral Phosphorus Ligands. .......................................................................................... 12 Figure 1.4 Phospholipid Bilayer. ...................................................................................................... 15 Figure 1.5 Phosphorus based Coenzymes. ................................................................................... 16 Figure 1.6 Energetic Phosphorous Molecules in Biological System. ................................ 17 Figure 1.7 Phosphorus Nerve Agents ............................................................................................. 18 Figure 1.8 Phosphorus Insecticides. ............................................................................................... 19 Figure 1.9 Phosphorus Based Herbicides. ................................................................................... 20 Figure 1.10 Fosfomycin and Other Natural Occurring Phosphonic Acid Antibiotics 21 Figure 1.11 Phosphorus Compound with Anticancer and Antiviral Activities. ........... 22 Figure 1.12 Bisphosphonates ............................................................................................................ 23 Figure 1.13 Phosphorus‐Based Hole Transporting Materials. ............................................ 24 Figure 2.1 Application of Aminophosphonates .................................................................. ……31 Figure 2.2 Fluorinated Drugs ............................................................................................................. 32 Figure 2.3 ORTEP Diagram of Diethyl 1‐fluoro‐2‐(4‐nitrophenylamino) propan‐2‐ ylphosphonate (Entry 2, in Table 1) ............................................................................................... 37 Figure 3.1 Deoxyribose Nucleic Acid and Purine and Pyrimidine Bases. ....................... 69 Figure 3.2 Two strands of DNA Showing Watson‐Crick Base Pairings. .......................... 70 Figure 3.3 Modified Nucleotide of Thymidine Derivatives. .................................................. 71 Figure 3.4 Nucleotide which Allows Universal Base. .............................................................. 72 x Figure 3.5 Transition State of the Active Site of DNA Polymerase Reactions. .............. 73 Figure 3.6 Two Molecules of H[(O 3 P) 2 CF 2 ] 3 ‐ Interconnected Through H‐Bond. ......... 78 Figure 3.7 Analogs of Triphosphonic Acids. ................................................................................ 80 Figure 3.8 Tritration Graph 3,3,3‐trifluoro‐2,2‐dihydroxypropylphosphonic Acid with 0.05 M NaOH Solution. ............................................................................................................... 84 Figure 4.1 Silyl‐enol Ether Type Intermediate from Sulfide and Sulfoxide ................. 115 xi List of Schemes Scheme 1.1 Deoxygenation of Phosphine Oxide with Trichlorosilane. .............................. 2 Scheme1.2 Nucleophilic Substitution Reaction of P(V) Compound..................................... 4 Scheme 1.3 Synthesis of Organophosphorus Compounds From White Phosphorus. .. 7 Scheme 1.4 A. Electrocatalytic Cycle of Ni Complexes. B. Results of Different Products Formed with Different Anodes. ........................................................................................ 8 Scheme 1.5 Wittig Olefination Reaction........................................................................................... 9 Scheme 1.6 Suggested Reaction Mechanism of Mitsunobu Reaction. .............................. 10 Scheme 1.7 Michaelis‐Arbuzov Reaction. ..................................................................................... 11 Scheme 1.8 Phosphorus Radical Based Deoxygenation Reaction ..................................... 13 Scheme 2.1 Kabanichk‐Fields Reaction ......................................................................................... 33 Scheme 2.2 Pathways for α‐Aminophosphonate Synthesis with Dialkylphosphites 34 Scheme 2.3 Possible Reaction Pathway for α‐Aminophosphonate with Triethyphosphite .................................................................................................................................... 38 Scheme 3.1 Synthesis of Difluromethyelenebisphosphonate. ............................................ 75 Scheme 3.2 Synthesis of Mono‐Flurobisphosphonate. ........................................................... 80 Scheme 3.3 Synthesis Ethylester of CF 2 Analog of Triphosphate. ..................................... 81 Scheme 3.4 Synthesis of Fluorinated Ethoxy(methyl)phosphoryl)methylphosphonate. ................................................................... 82 Scheme 3.5 Synthesis of 3,3,3‐Trifluoro‐2,2‐dihydroxypropylphosphonic .................. 83 Scheme 3.6 Synthesis Nucleotide Triphosphate Analogs. ..................................................... 85 Scheme 4.1 α‐Bromoacetophenone Derivatives as Potent PTP Inhibitors ................. 112 Scheme 4.2 Generation of NO 2 Cl in situ ...................................................................................... 112 xii Scheme 4.3 Ipso‐Nitration of Arylboronic Acids with in situ Generated ...................... 113 Scheme 4.4 TMSCl‐Nitrate Salt System as an Efficient Reagent for Oxidative Chlorination of Sulfides and Disulfides ....................................................................................... 114 Scheme 4.5 Plausible Mechanism for α‐Halogenation of Acetophenones with Negative Halide Species ..................................................................................................................... 119 Scheme 4.6 Plausible Mechanism for α‐Halogenation of Acetophenones with Positive Halide Species. ...................................................................................................................... 120 xiii Abstract This dissertation explores the field of organophosphorus chemistry. Phosphorus plays a major role in medicinal and natural product chemistry, which is the inspiration for the majority of the work presented in this dissertation. The first chapter is a short description of the vast field of organophosphorus chemistry. Phosphorus holds a unique place in the periodic table and its wide spread applications in various fields are presented. Short descriptions of the most important applications are explored, with special emphasis on phosphorus based reagents and biological relevant molecules. Chapter 2 describes the one pot synthesis of Kabachnik‐Fields reaction for fluorinated α‐aminophosphosphonates, amino acid analogs, with gallium triflate. Some mechanistic aspects are explored with respect to fluorinated imines and aminals. Chapter 3 deals with various pyrophosphonic acid analogs of bisphosphonic acids,triphosphate analogs. The pKa’s of 3,3,3‐trifluoro‐2,2‐ dihydroxypropylphosphonic acid, a pyrophosphonic acid analog are evaluated. The synthesis of modified nucleotide analog with 3,3,3‐trifluoro‐2,2‐ dihydroxypropylphosphonic acid is described. Chapter 4 presents a simple and mild reagent for the α‐chlorination and α‐ bromination of ketones with acidic α–hydrogens. TMS‐X (where X= Br,or Cl) and xiv KNO 3 salt is used to carry out α–bromination and α‐chlorination with good conversion and selectivity. 1 Chapter 1. Introduction to Phosphorus Chemistry 1.1 Characteristics of Phosphorus Compounds The importance of organophosphorus compounds is revealed in various forms in our daily lives. Organophosphorus compounds are used as agricultural chemicals, flame‐retardants, corrosion inhibitors, nanostructured materials, metal extractants, ligands for catalysis, in pharmaceuticals, and in various organic transformations as reagents and reaction mediators. 1,2,3,45,6,7,8,9,10,11,12,13 The chemistry of phosphorus continues to expand with its growing applications in various fields. Phosphorus in organic compounds can have diverse oxidation states ranging from P(I) low valent compounds to high valent P(V). The most common oxidation states of phosphorus in organophosphorus compounds are P(III) and P(V) compounds. P(III) compounds include phosphine and phosphites, and P(V) compounds include phosphoranes, phosphates, phosphonates, and phosphonic acids. P(III) compounds can readily undergo oxidation to P(V) in the presence of mild oxidants, thereby they can readily form phosphorus oxides (e.g. Ph 3 P=O) in the presence of air. The high bond dissociation energy of P=O (128‐139 kcal/mol 1 ) is often the driving force in determining the path of the reaction in phosphorus chemistry. The ease of formation of the P=O bond is the key factor in the Wittig olefination reaction and many other reactions, which will be discussed later in the chapter. 2 The P=O bond in most phosphorus compounds is short (1.452‐1.423Å), strong and highly polar, with a dipole moment of 4.51D (measured for Ph 3 P=O). 14 These properties account for the general characteristics exhibited by phosphine oxides during the course of various organic reactions. Phosphorus compounds containing a P=O group can generally form hydrogen bonds with water molecules and other protic solvents (depending on the nature of other substituents on phosphorus) and hence are readily soluble in these solvents. 1 The high bond strength of the P=O bond makes functionalization of compounds containing this functional group extremely difficult compared to its nitrogen counter part. Reduction of the P=O has been achieved with the recent developments of silane reagents. 15,16 Since oxygen‐silicon bond is significantly stronger than the P=O bond, the deoxygenation reaction is thermodynamically favorable. Trichlorosilane (Cl 3 SiH) is typically used in this process (Scheme 1.1) but other deoxygenation reagents have also been recently developed. 17,18,19 Scheme 1.1 Deoxygenation of Phosphine Oxide with Trichlorosilane. 3 Phosphorus can have coordination number as high as six in organophosphorus compounds. 1 Most phosphorus compounds have coordination numbers three and four are known, but during the last few decades a large number of compounds with coordination numbers of one, two, five and six at been discovered (Figure 1.1). The five‐coordinated peroxy compound (1.2) has been synthesized and can act as an oxidizing agent to form epoxides with various alkenes. 20 P R 1 O RO OH N N O O Me Me P O O _ OMe MeO MeO 1.1 1.2 Figure 1.1 Six and FiveCoordinate Phosphorus Compounds. Five‐coordinate P(V) compounds are considered to be involved in transition states and intermediate, in many nucleophilic substitution reactions at the P center. These transition state/intermediates have trigonal bipyramidal geometry, and longer bonds at the two axial positions compared to the three equatorial bonds. The position of the substituents in five coordinate phosphorus species are considered to be “fluxional” when functional groups attached to the phosphorus interchange their positions. This mechanism is referred to as Berry pseudorotation. 4 The “fluxional” characteristic of five‐coordinate phosphorus species can be observed in the nucleophilic substitution reaction of chiral P(V) compounds and has been extensively studied in phosphorus chemistry. The substitution usually takes places by nucleophilic attack at the axial position, generating a five‐coordinated phosphorus species. At this point the leaving group moves to the apical position as the bond lengths are longer in the axial position than at the equatorial positions (Scheme1.2), resulting in the formation of isomeric products. Scheme1.2 Nucleophilic Substitution Reaction of P(V) Compound. Phosphorus has a similar or and sometimes higher π electronegativity value than that of carbon, in terms of its ability; to accept and/or donate electron from its p orbital in low coordinate states. 21 The sigma electronegativity of carbon (2.5) however, is slightly higher than that of phosphorus (2.1). This finding can be explained with the apolar characteristic of the π component of the double bond in phosphaethene (HP=CH 2 ), whereas the σ component in phosphorus carbon ylides (P σ+ –C σ‐ ) is highly polarized. 22 This situation introduces some intriguing analogies between the reactivity of low‐coordinate derivatives of carbon and phosphorus in alkenes, and alkynes. 23 5 In phosphorylalkenes, the HOMO of the π‐bond is ‐10.3 eV and the lone pair is ‐10.7 eV. 24 where as, π ionization energy of C=C is around 10.51 eV. In P=C π‐ bond, the energy is calculated to be 43 kcal/mol compared to 65 kcal/mol for the C=C bond. 25,26 The conjugative properties are similar for P=C and C=C bonds. 27 A typical phosphaalkene has a P=C bond length of 1.60–1.70. 23 The P=C bond is generally highly reactive and cannot be observed under ordinary conditions unless some kind of stabilization is provided, by conjugation, steric hindrance, or complexation (Figure 1.2). P H Mes P OSiMe 3 Ph Ph P Ph P CH 2 Cl N N N Mes Mes W(CO) 5 1.3 1.4 1.5 1.6 Figure 1.2 Various Phosphoralkenes. Major developments in phosphorus chemistry have been enhanced by the development of NMR spectroscopy. Phosphorus has only one isotope with a molecular mass of 31 and spin of ½(dipolar nucleus), which makes it an ideal candidate for NMR spectroscopy. The 31 P NMR spectrum is highly informative; having a wide range of chemical shifts, from +300 ppm to ‐200 ppm, which can give detailed pictures of the molecular environment of phosphorus. The peak intensities observed in the 31 P NMR spectrum can be used to determine the quantitative ratio of different species in a reaction mixture containing 6 different phosphorus species. Hence, phosphorus NMR can be used for conformation studies as well as kinetic studies. Table 1.1 shows the range of 31 P chemical shifts observed in phosphorus compounds. Table 1.1 31 P NMR Shifts of Various Organophosphorus Compounds. The diverse chemistry of phosphorus compounds is attributed to the ability of phosphorus to form strong bonds with heteroatoms compared to its neighbor, 7 nitrogen. The bond dissociation energy for some of these elements, expressed in kcal/mols, are: P‐Cl =79, P‐Br= 63, P‐F =26, P‐O= 86 and P‐N= 55. Synthesis of organophosphorus compounds often starts with the elemental white phosphorus, P 4, in one or two steps (Scheme 1.3). The initial step involves direct chlorination of white phosphorus, followed by phosphorylation of organic substrates by phosphorus chloride, which produces large amounts of hydrochloric acid, making this process hazardous to the environment. Scheme 1.3 Synthesis of Organophosphorus Compounds From White Phosphorus. Recently Budnikova et. al developed an electrocatalytic method to generate triaryl phosphine from white phosphorus and organic halides using the transition metal complexes [Ni(bipy)]. 28,29,30 The electro‐catalytic cycle is shown in Figure 1.4 A. Nickel catalysts are generated at the cathode, and the catalyst generated in turn attack P4.30 The products of the electro‐catalytic cycle with Ni depend on the metals used at the anode. Products formed with different anodes are shown in Scheme 1.4 B 8 Scheme 1.4 A. Electrocatalytic Cycle of Ni Complexes. B. Results of Different Products Formed with Different Anodes. In another method, H 3 PO 4 , H 3 PO 3 and H 3 PO 2 were produced catalytically by solar irradiation of a suspension of white phosphorus in H 2 O/THF in the presence of water‐soluble ruthenium complexes containing mono‐ sulfonatedtriphenylphosphine. 31,32 These results highlight the potential of transition metals for producing organophosphorus compounds in an environmentally friendly process and such methods have gained much attention recently. 9 1.2 Phosphorus in Synthesis Plethoras of organophosphorus compounds have been used in various chemical transformations in organic synthesis. Phosphines, typically triarylphosphines, are the most commonly used reagents due to their good nucleophilic properties combined with the stability of the oxygen‐phosphorus double bond. These properties of phosphines are utilized in various organic transformations, with the Wittig olefination, one of the most well known example (Scheme1.5). There are innumerable examples of complex natural products, pharmaceutical, and materials that have been successfully synthesized using Wittig reagents. Scheme 1.5 Wittig Olefination Reaction. Triphenylphosphine is also used in the Mitsunobu reaction (Scheme 1.6). The Mitsunobu reaction is a versatile and widely used procedure for the synthesis of various acid derivatives. 33,34,35 This reaction involves the coupling of an alcohol with a acidic/pronucleophilic carbon to form esters, ethers, amides, etc., using a combination of a phosphine (which acts as a reducing agent), and an azo compound (which is an oxidizing agent). Mitsunobu reaction is generally highly stereoselective resulting in the inversion of configuration in secondary alcohols. Recently our 10 group has developed an efficient method for the stereoselective monofluorination of primary and secondary alcohols using Mistunobu reaction. 36 Scheme 1.6 Suggested Reaction Mechanism of Mitsunobu Reaction. One of the first developments in phosphorus chemistry was the discovery of the Michaelis‐Arbuzov 37,38 reaction where trialkylphosphites react with alkyl halides to form alkyl phosphonates (Scheme1.7). This reaction is one of the most effective ways to form P‐C bonds and is widely used to synthesize many useful organophosphorus compounds such as, phosphonates, phosphine esters and phosphine oxides. 39 Many starting material for the synthesis of phosphorus compounds are generated through this reaction and therefore it will be discussed in the following chapters. The mechanism for this reaction is widely debated and it is possible that the reaction proceeds through a single electron transfer process is operating. 11 R 1 O P OR 1 OR 1 R 2 X X=Halogen R 1 O P + O OR 1 R 2 R 1 X - R 1 X P O R 2 R 1 O OR 1 + Scheme 1.7 MichaelisArbuzov Reaction. Discovery by Knowles et. al. 40 of the application of chiral phosphines for asymmetric catalytic hydrogenation of olefins have made the synthesis of chiral phosphine ligands a much sought after field in organic synthesis. 41 Phosphorus (III) compounds are very good nucleophiles and good Lewis bacity, which makes them ideal ligands for metal complexation. 1 Phosphorus‐based chiral ligands are widely used in asymmetric catalysis for various transformations including hydrogenation, oxidation, cyclization and addition reactions. 42 The chirality of the phosphorus ligand can either originate from the scaffold to the phosphorus center, such as DIPAMP, 43 developed by Knowles, BINAP, developed by Noyori 44 or at phosphorus as used in the initial asymmetric hydrogenation reaction by Knowles. 40 12 PPh 2 PPh 2 N P N Me Me O N P P Ph Ph MeO MeO DIPAMP 1.7 1.8 1.9 BINAP Figure 1.3 Chiral Phosphorus Ligands. DIPAMP was one of the first commercially used diphosphine ligands for the synthesis of L‐DOPA, a prodrug for the treatment of Parkinson disease. 45 L‐DOPA was obtained in 95% ee after hydrogenation of the appropriate olefin precursor with DIPAMP, and is still the preferred way of synthesizing L‐DOPA. BINAP has also been used extensively as a chiral auxiliary. Recent advances in asymmetric catalysis have utilized the chiral Lewis‐base phosphorus compound (1.9, Figure 1.3)) in for various transformations including aldol reactions. 46 Since their initial discovery as possible intermediates in reactions, phosphorus radicals have been used as radical mediators as well as reagents. 47 P‐ centered radicals are very reactive towards unsaturated bonds and are mildly nucleophilic (they react faster with electron‐poor double bonds than electron rich double bonds). 48 Phosphorus radicals have been shown to have similar ability to abstract a halogen from alkyl halides as tin hydride. 49 Hence they can be used effectively as a mediator for reduction in place of tributyltin hydride. Although tin 13 compounds are extremely useful in reducing halides, their utilization has been limited due to their extreme toxicity and difficulties associated with removal of the by‐products from the reaction mixture. Phosphorus compounds also have been used as radical deoxygenation agents with AIBN as a radical initiator. 47 P‐based radicals were used in the deoxygenation of erthyromysic B derivates cleanly on a 15 kg scale (Scheme 1.8), for the industrial synthesis of ABT‐229, 50 which has been shown to be a potent motilin receptor agonist, 51 and have been tested in clinical trials as a potential prokinetic agent for the treatment of diabetic gastro paresis, gastro esophageal reflux disease, and functional dyspepsia. 52 Scheme 1.8 Phosphorus Radical Based Deoxygenation Reaction 1.3 Phosphorus Compounds in Biological System Phosphorus is a vital element in living organisms and is present in various organic and inorganic forms. The human body contains about 1% phosphorus by mass, about 80% of which is present as hydroxyapatite (Ca 5 (PO 4 ) 3 OH) in bones and 14 teeth. 53 The remaining phosphorus in living organisms is found as organic phosphates, phosphate esters or anhydrides due to the ability of phosphates to provide a binding handle for their derivatives. 54 Phosphate in the form of esters and anhydrides offer several potential modes of interaction with enzymes. These interactions include electrostatic interaction as in mono‐ and di‐anions, hydrogen bonding interaction both as donor‐acceptor or dipole‐dipole interactions. Hence, it is not surprising to see wide spread incorporation of phosphorus into biomolecules from metabolites to macromolecules. Table 1.2 shows the various uses of phosphorus containing molecules in biological systems. Table 1.2 The Biochemical Role of PhosphorusContaining Compounds 55 Phosphorus compounds play a crucial role in the storage and transmissions of genetic information in the form of nucleic acids and nucleotides. Phosphorus is also a key component of the cellular membrane in the form of phospholipids bilayer (Figure 1.4). Phospholipids contain a hydrophilic head consisting of phosphoric acid, and a hydrophobic tail consisting of fatty acids. The fatty‐acid moieties 15 aggregate to form the liquid bilayer, exposing the phosphoric acid moiety to the surface. Figure 1.4 Phospholipid Bilayer. Phosphoric acids and their esters are also present in many coenzymes (Figure 1.5) and play active roles in many metabolic and biosynthetic pathways. Most coenzymes based on phosphorus are derivatives of ribose sugars. Nicotinamide adenine dinucleotide (NAD + ) is a coenzyme involved in redox reactions in cellular metabolism and act as an electron source in the form of NADH. Cytidine triphosphate is used as coenzymes for the synthesis of phospholipids. Coenzyme A is involved in the metabolism of fatty acids and in pyruvate metabolism in the citric acid cycle. Adenosine triphosphate (ATP) is an efficient phosphate carrier in many enzymatic reactions functioning as a key chemical energy supplier. 16 Figure 1.5 Phosphorus based Coenzymes. Phosphorus‐containing cyclic nucleotide derivatives also play a significant role in the biochemical activity of diverse hormones, in the synaptic transmission of the nervous system, in cell regulation, and even in immune and inflammation response. Some phosphorus esters, such as myoinosotol 1,2,4‐triphosphate act as intercellular secondary messengers and control the calcium ion channels. Phosphoric acid derivatives are key energy suppliers for many biochemical pathways; among these, adenosine triphosphosphate (ATP) (Figure 1.6), also known as the currency of life, is the most common source of energy in many biochemical 17 pathways. Hydrolysis of ATP releases ‐7.3 kcal/mol of energy. 1 Phosphoenol pyruvic acid (PEP) 1.14 is the energy supplier for glycolysis and glucongenesis pathways and is the most energetic phosphate in living organisms, with an energy currency of ‐14 kcal/mol. Phosphophamidates (e.g. 1.15) also act as energy supplier for many biosynthetic pathways. Figure 1.6 Energetic Phosphorous Molecules in Biological System. Inorganic phosphoric acids also play a crucial role in biochemical processes, from being active carriers in cellular transport through cellular and mitochondrial membranes to bone metabolism. Due to the polyprotic nature, phosphoric acids also play an important role as an intracellular buffer system. 1.4 Phosphorous in Nerve Agents, Insecticides and Herbicides Nerve agents are classified as alkylphosphonic acid esters that contain at least one phosphorus‐carbon bond (Figure 1.7). Many functional groups containing phosphorus can be used to incorporate additional functionalities that contribute to 18 the unique properties of each individual member of nerve agents, such as persistence on surfaces, resistance to hydrolysis, solubility, and stability. 56 Figure 1.7 Phosphorus Nerve Agents Organophosphorus insecticides are relatively easy to synthesize and therefore can be modified to have a various range of toxicity by modifying substituents attached to phosphorus. The majority of phosphorus insecticides are based on thiophosphorus group, and a few have the phosphoryl group (Figure 1.8). Studies have shown that insecticides with an O‐methyl group have lesser toxicity to mammals compared to the O‐ethyl group. The greater interest towards organophosphorus insecticides has been due to their biodegradability. Phosphorus insecticides readily hydrolyze to phosphoric acids, minimizing the residual activity associated with chlorinated hydrocarbon pesticides. 19 Figure 1.8 Phosphorus Insecticides. Organophosphorus nerve agents were never used in chemical warfare, but their study has contributed enormously in the development of wide variety of insecticides and herbicides. Phosphorus‐based nerve agents and insecticides work with the same basic principle of inhibiting the activity of the enzyme acetylcholinesterase. 57 Acetylcholine is a neurotransmitter, which is involved in activating muscle contraction and regulating sodium ion channels in the nerve system. When acetylcholinesterase functions properly, the serine esterase hydrolyzes acetylcholine yielding acetate and choline and regenerating the active enzyme. Organophosphorus compounds can covalently block the active site of serine residue of acetylcholinesterase by undergoing nucleophilic attack to produce a serine–phosphoester adduct (Scheme 1.9). This irreversible inactivation leads to an excess of acetylcholine in the body and causes severe damage to the nervous system. 20 Scheme 1.9 Inhibition of Acetylcholine Enzymes by Organophosphorus Inhibitors. On the other hand herbicides have various modes of inhibition for bacterial and plant growth. Phosphinothricin (1.24) is a naturally occurring herbicide that inhibits the function of gultamine synthetase, which is important in bacteria and plant nitrogen metabolisms. 58 Glyphosate is a synthetic herbicide, which goes by the trade name Round‐up (1.25), it is a broad‐spectrum and non‐selective herbicide that is biodegradable and has low mammalian toxicity. Figure 1.9 Phosphorus Based Herbicides. 1.5 Organophosphorus Compound in Medicine One of the initial discoveries of phosphorus compounds in medicine was the finding of Fosfomycin, a phosphonic acid derivative that is produced from the fermentation broth of the bacterium Streptomyces Fradiae. 59 Soon, other phosphonic acids were also abstracted from bacterial broths showing antibacterial activities 21 (Figure 1.10). Fosfomycin (1.26) is active towards both gram positive and gram negative bacteria and its effectiveness is comparable with that of the well‐known antibiotics. Figure 1.10 Fosfomycin and Other Natural Occurring Phosphonic Acid Antibiotics This discovery opened up a whole new field of research in phosphorus compounds with P‐C bonds in medicinal chemistry. Phosphorous antibiotics are relatively simple with easily accessible structures compared to the highly complex structures of most antibiotics. Phosphorus compounds of diverse structures have been shown to posses high levels of anticancer and antiviral activities. Cyclophosphamide (1.29) is a prodrug, which is widely used as an anticancer agent. Fascarnet (1.30) is known to inhibit DNA polymerase and is used for the treatment of Herpes Simplex virus and also shows significant activity against HIV. Large efforts are currently being made in this area for rational synthesis of drugs against HIV and cancer. 22 Figure 1.11 Phosphorus Compound with Anticancer and Antiviral Activities. Bisphosphonates are the non‐hydrolyzable form of pyrophosphate, where the oxygen linker between two phosphorus atoms (P‐O‐P) is replaced by carbon (P‐ C‐P). Studies have shown that pyrophosphates inhibit the formation and dissolution of hydroxyapatite (Ca 5 (PO 4 ) 3 OH) in bone, preventing calcification of tissues by maintaining bone minerals. Hence, bisphosphonates have gained much attention as pharmaceutical agents being isosteric to pyrophosphate, with the added advantage of being hydrolytically stable, and can be used in the treatment of various bone deteriorating diseases including osteoporosis and Paget diseases. 60,61 Etidronateis (1.31) and Chloronate (1.32) were the first bisphosphonates used in humans for the treatment of Paget disease and osteoporosis, but recently derivatives containing alkylamino side chain substituents are gaining much attention (1.33, Figure1.12) Bisphosphonates as pharmaceutical agents are being used as antidepressant (1.34) and anti‐hypercholesterolemia agents (1.35). Recently there has been intense research in bisphosphonate chemistry for synthesizing modified nucleotides, which is a key research topic in this thesis and 23 will be further discussed in Chapter 3. Bisphosphonates are also used as fertilizers, in textile and other industries. 62 Figure 1.12 Bisphosphonates 1.6 Phosphorus in Materials. Some of the most important flame‐retardant material on the market are organophosphorus compounds. Most phosphorus‐based flame retardants function by forming phosphoric or polyphosphoric acids which absorb water from the surroundings and accelerate the formations of chars. Phosphorus‐based materials are used in the electronic industries, such as in organic light emitting diodes (OLED). In its simplest form OLEDs consist of a hole transporting layer, which is a p‐type semiconductor, an electron transporting layer which is an n‐type semiconductor, and a spacer layer. 63 The hole transporting layer usually consists of tertiary amines with aromatic substituents. 64 Organophosphorus compounds (Figure1.13) have been recently introduced in OLED, for hole 24 transporting layers due to their structural similarities with their nitrogen counter parts, as well as due to their high thermal stability compared to nitrogen‐based compounds. Thermal properties of these compounds are of particular interest since high T g and thermal stabilities are needed to withstand high temperature encountered during the OLED operation. Figure 1.13 PhosphorusBased Hole Transporting Materials. 1.7 Conclusions The field of organophosphorus chemistry is quite mature, with the growing application in pharmaceutical and in material industries. Phosphorus holds a unique place in the periodic table, as evidenced by the diverse structural and chemical properties displayed by the variety of organophosphorus compounds. It is however possible to fully describe the tremendous impact that phosphorus compounds have made in various fields in this short introductory chapter. I have tried to focus on areas where phosphorus is dominant, especially in biological systems and pharmaceuticals and in some new emerging areas where much more development can be envisioned. 25 1.8 Chapter 1 References 1. Quin, L. D. A Guide to Organophosphorus Chemistry; Wiley‐Interscience: New York, 2000. 2. Dillon K. B.; Mathey, F.; Nixon J.F. Phosphorus: The Carbon Copy; John Wiley & Sons, Inc.: New York, 1998. 3. Johndon, S. E. Ylides and Imines of Phosphorus; John Wiley & Sons, Inc.: New York, 1993. 4. Engel, R. Handbook of Organophosphorus Chemistry; Marcel Dekker, Inc.: New York, 1992. 5. Regitz, M.; Scherer, O.J., eds Multiple Bonds and Low Coordination in Phosphorus Chemistry; Georg Thieme Verlag; Stuttgart, Germany, 1990. 6. Hartley, F. R. The Chemistry of Organophosphorus Compounds; John Wiley & Sons, Inc.: New York, 1990; Vol 2. 7. Toy, A. D. F.; Walsh, E. N. Phosphorus Chemistry in Everyday Living; 2nd ed.; American Chemical Society: Washington D.C., 1987. 8. Hilderbrand, R.L. The Role of Phosphonates in Living Systems; CRC Press: Boca Raton. 1983. 9. Hori, T.; Horiguchi, M., Hayashi, A. Biochemistry of Natural CP Compounds; Japan Association for Research on the Biochemistry of C‐P Compounds; Maruzen, Japan, 1984. 10. Cadogan, J.L.G.; Editor Organophosphorus Reagents in Organic Synthesis, 1979. 11. McEwen, W.E.; Berlin, K. D., eds Organophosphorus Stereochemistry; Halsted Press: New York, 1975. 12. Kirby, A. J.; Warren, S. G. The Organic Chemistry of Phosphorus; Elsevier: Amsterdam, 1967. 13. Hudson, R.F. Structure and Mechanism in OrganoPhosphorus Chemistry; Academic Press : New York, 1965. 14. F. R. Hartley, ed., The Chemistry of Organophosphorus Compounds, Vol. 2. John Wiley & Sons, Inc., New York, 1990 26 15. Fritzsche, H.; Hasserodt. U; Koerw. F., Chem. Ber. 1964, 97, 1988. 16. Organic Synthesis, Coll. Vol. 1993. III. 57 17. Zablocka, M.; Delest, B.; Igau, A. Skowronska, A.; Majoral, J‐P., Tetrahedron Lett., 1997, 38, 5667 18. Lawerense, N. J.; Muhammad, F., Tetrahedron, 1998, 54, 15361. 19. Griffin, S.; Heath, L.; Wyatt, P., Tetrahedron Lett., 1998, 39, 4405. 20. Ho, David G.; Gao, R.; Celaje, J.; Chung, H.; Selke, M., Science, 2003, 302, 259‐262. 21. Waluk, J.; Klein, H.‐P.; Ashe III, A. J.; Michl, J. Organometallics 1989, 8, 2804. 22. Schoeller,W. W., Chem. Commun., 1985, 334. 23. Mathey, F., Angew. Chem. Int. Ed., 2003, 42, 1578 – 1604. 24. Locombe, S.; Gonbeau, D.; Cabioch, J.‐l; Pellerin, B.; Denis, J.‐M.; Pfister‐Guillouzo, G. J. Am. Chem. Soc., 1988, 110, 6964. 25. Schmidt, M.W.; Truong, P. N.; Gordon M. S. J. Am. Chem. Soc., 1987, 109, 5217. 26 Schleyer, P. von R. ; Kost, D. J. Am. Chem. Soc., 1988, 110, 2105. 27. Nyulaszi, L.; VeszprTmi, T.; RTffy J. J. Phys. Chem., 1993, 97,4011. 28. Budnikova,Y. H.; Yakhvarov, D. G.; Sinyashin, O. G. J. Organomet. Chem., 2005,690, 2416. 29. Budnikova,Y. H.; Yakhvarov, D. G.; Kargin, Y. M. Mendeleev Commun., 1997, 67– 68 30. Budnikova,Y. H.; Perichon, J.; Yakhvarov, D. G.; Kargin, Y. M.; Sinyashin, O. G. J. Organomet. Chem., 2001, 630, 185. 31. Peruzzini, M,; Gonsalvi, L; Romerosa, A. Chem. Soc. Rev., 2005, 34, 1038–1047. 32. Romerosa, A.; Manãs S.; Richter, C. Spanish Patent,P200201731 A1 2209628 B2 2209628. 27 33. Mitsunobu, O. Synthesis, 1981, 1. 34. Hughes, D. L. Org. React.,1992, 42, 335. 35. Hughes, D. L. Org. Prep. Proced. Int., 1996, 28, 127. 36. Prakash, G.K.S.; Chacko, S; Alconcel, S.; Stewart,T.; Mathew, T.G.; Olah, G.A. Angew. Chem. Int. Ed., 2007, 46, 4933. 37. Michaelis, A.; Kaehne, R.;Ber. Dtsh. Chem. Ges., 1898, 31, 1048. 38. Arbuzov, R. A. Pure Appl. Chem., 1964, 9, 307. 39. Bhattacharya, A.K.; Thyagarajan G, Chem. Rev., 1981, 81, 415. 40. Knowles, W. S.; Sabacky, M. J. Chem. Commun., 1968, 1445 41. Kagan, H. B.; Sasaki, M F. R , Hartely, ed. The Chemistry of Organophosphorus Compounds, Vol. 1. John Wiley & Sons, Inc., New York, 1990, Chapter 3. 42. Ojima, L. Catalytic Assymetric Synthesis; 2nd ed.; John Wiley & Son, Inc.: New York, 2000 43. Vineyard, B. D.; Knowles,W. S.; Sabacky, M. J.; Bachmanand, G. L.; Weinkauff, D. J., J. Am. Chem. Soc., 1977, 99, 5946. 44. Miyashita, A.; Yasuda,A.; Takaya, H.; Toriumi, K.; Ito, T.; Souchi, T.; Noyori, R. J. Am. Chem. Soc., 1980, 102, 7932. 45. Eberhardt, L.; Armspach, D.; Harrowfield, J. Matt, D., Chem. Soc. Rev., 2008, 37, 839. 46. Denmark, S. E.; Stavenger, R. A. Acc. Chem. Res., 2000, 33 432 47. Leca, D.; Fensterbank, L.; Lacôte, E.; Malacria, M. Chem. Soc. Rev., 2005, 34, 858. 48. Sumiyoshi, T.; Schnabel,W.; Henne, A.; Lechtken,P. Polymer, 1985, 26, 141 49. Jang, D. O. Tetrahedron Lett., 1996, 37, 5367. 50. Graham, A. E.; Thomas, A. V.; Yang, R. J. Org. Chem., 2000, 65, 2583. 28 51. Lartey, P. A.; Nellans, N. H.; Faghih, R.; Petersen, A.; Edwards, C. M.; Freiberg, L.; Quigley, S.; Marsh, K.; Klein, L. L.; Plattner, J. J. J. Med. Chem., 1995, 38, 1793. 52. Omura, S.; Tsuzuki, K.; Sunazuka, T.; Marui, S.; Toyoda, H.;Inatomi, N.; Itoh, Z. J. Med. Chem., 1987, 30, 1941 53. Organophosphorus chemistry Goldwhite, H. Introduction to Phosphorus Chemistry; CambridgeUniversity Press: Cambridge, U.K., 1981. 54. Berkowitz, D. B.; Bose, M. J. Fluor. Chem., 2001, 112, 13. 55. Maciá, E. Chem. Soc. Rev., 2005, 34, 691. 56. Eubanks L. M.; Dickerson, T. J.; Janda, K. D., Chem. Soc. Rev., 2007, 36, 458. 57. Singh, B. K.; Walker, A. FEMS Microbiol. Rev., 2006, 30, 428. 58. Harmmerschmidt, F.; Kachlig. H. J. Org. Chem., 1991, 56, 2364. 59. D. Hendlin, et al., Science 1969,166, 122 60. Fleisch, H. A. Drugs, 1991, 42, 919. 61. Bijvoet, O. L. M.; Fleisch, H. A.; Canfield, R. E.; Russell, R. G. G., Eds., Bisphosphonates on Bones, Elsevier, Amsterdam, 1995 62. Blomen, L. J. M. J. Bisphosphonates on Bones 1995, 111. 63.Borek, C. PhD. Thesis, University of Southern California, 2008. 64. Baumgartner, T.; Rau, R. Chem. Rev., 2006, 106, 4681. 30 Chapter 2. Synthesis of Fluorinated αAminophosphonates using Gallium Triflate as catalyst. 2.1 Introduction The significance of α‐amino acids in biological system, as structural units in peptides, proteins, and enzymes has led to intense interest in the chemistry and biological activities of their synthetic analogues. Phosphorus analogues of amino acids, in which the carboxylic group is replaced by a phosphonic, P(O)(OH) 2 , or phosphinic acid group, P(O)(OH)R, as well as a phosphonate group, P(O)(OR) 2 have great potential, particularly in the preparation of isosteric or bioisosteric analogues of numerous natural products. As mimics of the natural α‐amino acids, α‐ aminophosphonic acids and its esters can act as enzyme and protease inhibitors 1,2,3,4 and have regulatory effect on plant‐growth, 5 act as herbicides, 6 and can also function as carriers of charged molecules across cellular membranes. 7 Some α‐ aminophosphonates are shown to exhibit anti‐HIV, 8 antibacterial 9 and neuronal activities. 10 The replacement of the carboxylic group by phosphonic acid moiety has a number of significant consequences in terms of shape, acidity and steric bulkiness. Phosphonic acids are tetrahedral, have increased acidities (R–PO 3 H 2 , pKa 2.5, 8.0) 11 and are bulkier than the natural amino acids containing carboxylic derivatives. This results in sufficient structural modification for these amino acid analogues to inhibit enzymes or receptors to which normal amino acids could easily interact. Hence α‐ 31 aminophosphonic acid is also antagonist to natural amino acids. Some of the important applications of α‐aminophosphonate in various fields are depicted in Figure 2.1. Figure 2.1 Application of Aminophosphonates In recent years, there has been instense activity in the developmentof various fluorinated drugs are dominant in the pharmaceutical industry (Figure 2.2). Fluorinated nucleosides, alkaloids, macrolides, steroids, and sugars are used as therapeutic agents showing anticancer, antiviral, antibiotic and anti‐inflammatory agents. 12 Fluorine can act as hydrogen mimics at enzyme receptor sites due to its steric compatibility with hydrogen. 13 It can significantly alter the chemical reactivity of a drug, due to its high electronegativity. The high electronegativity has significant effect on the acidity and basicity of its neighboring functional groups, which in turns affects the metabolic stability of a fluorinated compound. The strong carbon‐ 32 fluorine bond in fluorinated pharmaceuticals also contributes to better thermal and oxidative stability. The overall basicity, lipophilicity, hydrogen bonding is greatly altered when fluorine is incorporated in molecules, and this contributes to the metabolic stability and therefore bioavailability of therapeutic drugs in biological system. Hence, fluorinated α‐aminophosphonates would be great interest in biological system. Figure 2.2 Fluorinated Drugs 33 2.2 Results and Discussions There have been numerous methods for generating α‐aminophosphonate framework, [N‐C‐P(O)], including two and three steps processes, 14 but the most efficient method to date is the one‐pot three component Kabachnik‐Fields reaction, where the α‐aminophosphonates are formed from amines (ammonia), ketones or aldehydes, and dialkylphosphites (Scheme 2.1). 15 Scheme 2.1 KabanichkFields Reaction The mechanism of the Kabachnik‐Fields reaction largely depends on the substituents on the substrates employed in the process. The pathways by which the Kabachinik‐Fields reaction can take place are shown in Scheme 2.2, including the two‐and three steps reaction pathways. The Kabachnik‐Fields reaction can either take place through an aminal intermediate (2.5) or through the Pudovik reaction, where dialkylphosphite can react initially with imines or ketones/aldehydes (Scheme 2.2). 34 Scheme 2.2 Pathways for αAminophosphonate Synthesis with Dialkylphosphites Various catalysts have been utilized in the synthesis of Kabachnik‐Fields reaction, including Lewis‐and Brønsted‐acid catalysts. 16,17,18,19,20,21,22,23,24,25,26,27 The Pudovic reaction pathway usually employs either a base, 28,29,30 which initially deprotonates the dialkylphosphite, or Lewis acids, 31,32,33,34,35,36 which catalyze imine formation as well as the dialkylphosphite addition with the imine. Most catalysts used in these processes are toxic, and cannot be recovered from the reaction mixtures. Gallium triflate is a versatile Lewis Acid catalyst for various transformations in organic synthesis. 37,38,39,40,41,42,43,44,45,46 The Gallium triflate is water soluble and is easily accessible and thermally stable upto 280 °C. Studies in our group have shown that gallium (III) triflate is an effective catalyst that is mild as 35 well as non‐hydrolysable in aqueous medium and can be easily recovered from the reaction mixture, making it an attractive green catalyst. 47 Numerous fluoroorganics of pharmaceutical interest have been synthesized by our group and others. Among these, synthesis of fluorinated α‐amino‐acids derivatives from fluorinated ketones by a three component approach (Strecker reaction) is of great interest, due to the lack of any other convenient methods. Gallium triflate offers optimum acidity required for general ketonic Strecker reaction which has remained a challenge until recently. 37 This prompted us to utilize gallium triflate as a catalyst for the synthesis of fluorinated α‐aminophosphonates. We carried out the Kabachnik‐Fields reaction using 5 mol% of gallium triflate with an equimolar amount of amine, fluorinated acetone and triethylphosphite in dry THF in a pressure tube at room temperature. The results are shown in Table 2.1. 36 Table 2.1 Results of the KabachnikFields Reaction for Fluorinated α Aminophosphonates When the reaction was monitored by TLC, formation of mixture of products was observed. The reactions yielded multiple unidentified products as determined by thin‐layer chromatography (TLC). Significant amounts of N‐ethyl‐aniline derivative were also observed in the reaction mixture. The x‐ray crystal structure of diethyl 1‐fluoro‐2‐(4‐nitrophenylamino) propan‐2‐ylphosphonate (Entry 2, Table 37 2.1, Figure 2.3) was also obtained. p‐Nitro‐aniline was observed to give better yield of the isolated products compared to other substituted anilines. We were only able to isolate p‐nitro‐aniline substituent of α‐ aminophosphonates with difluroacetone (Entry 1, Table 1). We were not able obtain α‐aminophosphonates with trifluoroacetone. The reaction also did not proceed to the desired products when other alkylamines were used as the reactants. The yields ranged from 10‐20%, compared to the literature yields 80‐90% for other α‐ aminophosphonates using other catalysts. Figure 2.3 ORTEP Diagram of Diethyl 1fluoro2(4nitrophenylamino) propan2ylphosphonate (Entry 2, in Table 1) As mentioned previously, the Kabachnik‐Field reactions are usually carried out using dialkylphosphites. There are very few examples wherein trialkylphosphites were used. 24 In our studies, we used trialkylphosphites as the 38 reactant because trialkylphosphites are more nucleophilic than dialkylphosphites and can readily undergo oxidation to phosphonates in the presence of a mild oxidant due to the stability of P=O (See Chapter 1). Trialkylphosphites can attack fluorinated acetone (fluorinated carbonyl being highly electrophilic) by Pudovic reaction to produce a hydrophosphonate (2.7), which can readily, irreversibly rearranges to the phosphonates (2.8). This rearrangement product is attributed to the low yield of α‐aminophosphonate. Both hydroxyphosphonate (2.7) and the phosphonates (2.8) were identified by 31 P NMR spectroscopy. Scheme 2.3 Possible Reaction Pathway for αAminophosphonate with Triethyphosphite When the reaction was conducted at low temperature (‐78°C to 0°C), and monitored using 31 P NMR spectroscopy, it was found that no reaction occurred at 39 these temperature. However, upon warming the reaction mixture to room temperature, the Pudovic reaction product was formed. The decline in the α‐ aminophosphonate product, by the competing influence of Pudovic product was further confirmed by performing a controlled reaction was carried out using triethylphosphite and trifluoroacetophenone. The Pudovic reaction product was obtained almost exclusively within minutes after mixing the reactants at room temperature without the use of catalysts. Table 2.2 Result for Imine Formation Under Conventional Heating To test the imine reaction pathway, we generated fluorinated imines using gallium triflate catalyst and other acid catalysts such as SAC‐13 and montmorillonite K‐10. Under conventional heating, using toluene as the solvent, gallium triflate produced the best results (Table 2.2). We also tested the formation of fluorinated 40 imines under microwave irradiation (Table 2.3). Microwave reactions were carried out at 165 °C without any solvent using these three catalysts and it was found that montmorillonite K‐10 gave much better yield of imines than other ones. Table 2.3 Result for Imine Formation Under Microwave Conditions When the imines from these reaction mixtures were tested with triethylphosphite under various conditions, α‐aminophosphosphate did not form in the presence of these catalysts. Hence, it is possible that α‐aminophosphosphates formed through the aminal intermediates. Therefore it is clear that the Pudovic reaction dominates the reaction pathways in this one‐pot Kabachnik‐Fields reaction, which results in the low yield of the desired fluorine containing α‐ aminophosphonates. 2. 3 Conclusions We have synthesized and characterized new fluorinated analogs of α‐ aminophosphonates using gallium triflate. The yields are relatively low compared to 41 other methods presented in literature for the synthesis of α‐aminophosphonates due to rapid formation of hydroxyphosphonates, which rearranges to phosphonates 2.4 as shown in Scheme 2.2 Our group has been involved in synthesis of pyrophosphate analogs of for modified nucleotides to study the structural‐activity relationship (SAR) of Pol β‐ DNA polymerase. These fluorinated α‐aminophosphonates analogues can potentially be used in designing new class of modified nucleotides. This will be further discussed in the following chapter. 2.4 Experimental 2.4.1 General Unless otherwise mentioned, all chemicals were purchased from commercial sources. THF was dried over sodium under nitrogen atmosphere. 1 H, 13 C, 19 F , and 31 P NMR spectra were recorded on a Varian Mercury series 400MHz NMR spectrometer 1 HNMR chemical shifts were determined relative to internal tetramethylsilane, at δ 0.0 or from the residual solvent peaks. 13 C NMR chemical shifts were determined relative to internal tetramethylsilane, at δ 0.0 , or to the 13 C signal of CDCl 3 at δ 77.0. 19 F NMR chemical shifts were determined relative to an external standard CFCl 3 at δ 0.0. 31 P NMR chemical shifts were determined with an external standard H 3 PO 4 , at δ 0.0. Microwave reaction was carried out using a Biotage apparatus. 42 2.4.2 Typical synthesis of αAminophosphonates In a pressure tube equimolar (1mmol) amount of aryl amine, fluorinated acetone, triethylphosphite were dissolved in 10 mL of dry THF and the mixture stirred for 2 hrs. The reaction progress was monitored by 31 P NMR spectroscopy. The reaction mixture was transferred to a round bottom flask and THF removed under reduced pressure. The residue was dissolved in 50 mL of methylene chloride and washed thrice with 10 mL of water. The organic layer was dried with anhydrous MgSO 4 , filtered and the solvent removed under reduced pressure. The product was separated using preparative TLC with a mixture (1:2:7) of ethylacetate, methylene chloride, and hexane, respectively. 2.5 Spectral Data: Diethyl 1,1difluoro2(4nitrophenylamino)propan2ylphosphonate H N (EtO) 2 P O NO 2 HF 2 C CH 3 1 H‐NMR δ 1.33 (t, 6H, 3 J=7.1Hz), 1.69 (d, 3H, 3 J P‐H =15.3Hz), 4.20 (m, 4H), 4.69 (d, 1H, J=8.0Hz), 6.11 (dt, 1H, 3 J P‐H =1.5Hz, 2 J F‐H =55.0Hz), 7.00 (d, 2H, 3 J H‐H =9.2Hz), 8.09 (d, 2H, 3 J H‐H =9.2Hz). 13 C δ 16.231(d, 2C), 16.641(d, 1C), 63.432(d, 2C), 113.56 (s 1C) 116.72 (s 2C), 125.80 (s, 2C), 126.55 (s 1C) 150.68 (s, 1C). 19 F‐NMR δ ‐127.89 (dddd, 1F, 3 J P‐F =12.1Hz, 2 J F‐H =54.9Hz, 2 J F‐H =71.2Hz, 2 J F‐F =279.1Hz) 31 P‐NMR δ 20.96 (dd, 1P, 43 3 J P‐F =8.4Hz, 3 J P‐F =15.9Hz) HRMS: m/z [M+Na+] calcd for C 13 H 19 F 2 N 2 O 5 PNa: 375; found: 375.0886. Yield 10% Diethyl 1fluoro2(phenylamino)propan2ylphosphonate Yellowish oily liquid. 1H‐NMR δ 1.27 (m, 6H) 1.41 (d, 3H, 4 J H‐H =2.3Hz, 3 J P‐H =15.9Hz), 4.12 (m, 4H), 4.39 (m, 1H), 4.65 (m 1H), 6.92 (m, 2H), 6.99 (m, 2H), 7.18 (m, 1H). 13 C‐NMR δ 16.63 (m, 1C), 17.55 (m, 2C), 58.41 (dd,1C 3 J F‐C =17.8Hz, 1 J P‐C =155.7Hz), 63.04 (m, 2C), 85.05 (m, 1C) 122.21 (s, 2C) 122.46 (s, 2C) ppm 129.02 (s, 1C) 143.98 (m, 1C). 19 F‐NMR δ ‐224.85 (dt, 1F, 3 J F‐P =14.1Hz, 1 J F‐H =47.5Hz). 31 P‐NMR δ 25.97 (d, 1P 3 J P‐F =14.1Hz). HRMS: m/z [M+Na+] calcd for C 13 H 21 NO 4 PNa: 312.1135 found: 312.1135. Yield 10% Diethyl 1fluoro2(4nitrophenylamino)propan2ylphosphonate H N (EtO) 2 P O NO 2 FH 2 C CH 3 Yellow solid, 1 H‐NMR δ 1.29 (m, 6H), 1.60 (dd, 3H, 3 J F‐H =2.1Hz, 2 J P‐H =15.1Hz), 4.13 (m, 1H), 4.53 (ddd, 1H, J=9.7Hz, J=14.1Hz, J=47.5Hz) 4.66 (d, 1H, J=7.7Hz), 4.78 (ddd, 1H, J=5.8Hz, J=9.7Hz, J=47.5Hz), 6.97 (d, 2H, 3 J H‐H =9.3Hz), 8.05 (d, 2H, 3 J H‐H =9.2Hz). 44 19 F NMR δ ‐226.81 (dt, 1F, 3 J F‐P =17.0Hz, 2 J F‐H =48.8Hz). 13 C‐NMR δ 16.78 (m, 2C), 17.96 (d, 1C, 2 J P‐C =5.7Hz), 57.88 (dd, 1C, 2 J C‐F =17.6Hz, 1 J P‐C =156.3Hz), 63.58 (m, 2C), 85.35 (dd, 1C, 2 J C‐P =5.3Hz, 1 J C‐F =181.6Hz) 31 P NMR δ 24.08 (d, 1P 3 J P‐F = 17.54), 19 F NMR δ HRMS: m/z [M+Na+]calcd for C 13 H 20 FN 2 O 5 PNa: 357.0986.; found: 357.0981. Yield 20% Diethyl 1fluoro2(ptolylamino)propan2ylphosphonate H N (EtO) 2 P O FH 2 C CH 3 Dark yellow liquid, 1 H‐NMR δ 1.27 (m, 1H), 1.41 (dd, 3H, 3 J F‐H =2.3Hz, 2 J P‐H =16.0Hz), 2.26 (s, 3H), 4.13 (m, 1H), 4.39 (m, 1H), 4.64 (m, 1H), 6.78 (dd, 2H, 3 J H‐H =20.5Hz), 7.06 (dd, 2H, 3 J H‐H =11.0Hz); 13 C‐NMR δ 16.64 (m, 1C), 21.66 (s, 1C), 63.01 (m, 2C), 84.98 (d, 2C, J=174.4Hz), 119.57 (s, 1C), 123.19 (d, 1C, J=19.5Hz), 128.84 (s, 1C), 138.79 (s, 1C), 143.88 (s, 1C); 31 P‐NMR δ 26.01 (d, 3 J P‐F =13.8Hz). HRMS: m/z [M+Na+] calcd for C 14 H 28 NO 3 PNa: 326.1292.; found: 326.1292. Yield 10% Diethyl 1fluoro2(2fluorophenylamino)propan2ylphosphonate H N (EtO) 2 P O FH 2 C CH 3 F 45 1 H‐NMR δ 1.28 (m, 6H), 1.47 (dd, 3H, 3 J F‐H =2.1Hz, 2 J P‐H =15.7Hz), 4.14 (m, 1H, 4.46 (m, 1H), 4.70 (m, 1H), 6.82 (m, 1H), 6.97 (m, 1H), 7.30 (m,1H). 13 C‐NMR δ 16.62 (m, 1C), 17.59 (d, 1C, 1 J C‐C =6.0Hz), 58.14 (m, 1C), 85.18 (dd, 1C, 1 J C‐C =5.4Hz, 1 J C‐ F =179.4Hz), 115.17 (d, 1C, 1 J C‐C =20.5Hz), 121.65 (d, 1C, 1 J C‐C =7.4Hz), 122.28 (s, 1C), 124.26 (d, 1C, 1 J C‐C =3.8Hz), 132.52 (m, 1C), 154.96 (d, 1C, 1 J C‐F =240.2Hz). 19 F‐NMR δ ‐226.37 (dt, 1F, 3 J P‐F =17.4Hz, 2 J F‐H =49.1Hz), ‐131.35 (s, 1F). 31 P‐NMR δ (d, 1P, 3 J P‐F =13.8Hz). Yield 10% 2.6 Crystal Structure Data Table 2.4 Crystal data and structure refinement for C 13 H 17 FN 2 O 5 P Identification code pfm Empirical formula C 13 H 17 F N 2 O 5 P Formula weight 331.26 Temperature 133(2) K Wavelength 0.71073 Å Crystal system Monoclinic Space group P2(1)/c Unit cell dimensions a = 8.994(4) Å α= 90°. b = 9.661(5) Å β= 92.864(8)°. c = 18.425(9) Å γ = 90°. Volume 1599.1(13) Å 3 Z 4 Density (calculated) 1.376 Mg/m 3 Absorption coefficient 0.206 mm ‐1 F(000) 692 Crystal size 0.54 x 0.30 x 0.08 mm 3 Theta range for data collection 2.21 to 27.53°. Index ranges ‐11<=h<=11, ‐8<=k<=12, ‐23<=l<=18 Reflections collected 10399 46 Table 2.4 Continued Independent reflections 3557 [R(int) = 0.0376] Completeness to theta = 27.53° 96.5 % Absorption correction Semi‐empirical Max. and min. transmission 0.9833 and 0.8964 Refinement method Full‐matrix least‐squares on F 2 Data / restraints / parameters 3557 / 0 / 201 Goodness‐of‐fit on F 2 1.052 Final R indices [I>2sigma(I)] R1 = 0.0877, wR2 = 0.2628 R indices (all data) R1 = 0.1221, wR2 = 0.2914 Largest diff. peak and hole 1.538 and ‐0.668 e.Å ‐3 47 Table 2.5 Atomic Coordinates ( x 10 4 ) and Equivalent Isotropic Displacement Parameters (Å 2 x 10 3 )for C 13 H 17 FN 2 O 5 P. U(eq) is Defined as One Third of the Trace of the Orthogonalized U ij Tensor _______________________________________________________________________________________ x y z U(eq) ________________________________________________________________________________________ P(1) 10595(1) 1997(1) 1065(1) 54(1) F(1) 6810(5) 3556(4) 133(2) 116(1) N(1) 8128(3) 887(3) 376(2) 45(1) N(2) 3721(4) ‐1468(4) 2033(2) 52(1) O(1) 11504(3) 770(4) 932(2) 66(1) O(2) 9989(3) 2107(4) 1839(2) 67(1) O(3) 11363(4) 3445(5) 944(2) 92(1) O(4) 3421(4) ‐922(4) 2614(2) 67(1) O(5) 3066(4) ‐2488(4) 1792(2) 65(1) C(1) 8967(4) 2185(4) 430(2) 46(1) C(2) 9560(5) 2417(5) ‐336(2) 60(1) C(3) 8024(5) 3435(5) 625(3) 57(1) C(4) 10364(6) 1171(6) 2444(3) 73(1) C(5) 11414(9) 1829(7) 2974(3) 95(2) C(6) 12728(10) 3880(12) 1325(4) 174(5) C(7) 13896(8) 3991(12) 976(5) 152(4) C(8) 7085(4) 356(4) 819(2) 40(1) C(9) 6750(4) 882(4) 1507(2) 44(1) C(10) 5658(4) 277(4) 1897(2) 46(1) C(11) 4879(4) ‐853(4) 1621(2) 43(1) C(12) 5206(4) ‐1411(4) 953(2) 46(1) C(13) 6291(4) ‐820(4) 561(2) 44(1) _________________________________________________________________________________________ 48 Table 2.6 Bond lengths [Å] and angles [°] for C 13 H 17 FN 2 O 5 P ____________________________________________________ P(1)‐O(1) 1.468(4) P(1)‐O(2) 1.555(3) P(1)‐O(3) 1.581(4) P(1)‐C(1) 1.837(4) F(1)‐C(3) 1.389(6) N(1)‐C(8) 1.374(5) N(1)‐C(1) 1.465(5) N(2)‐O(5) 1.220(5) N(2)‐O(4) 1.236(5) N(2)‐C(11) 1.447(5) O(2)‐C(4) 1.462(6) O(3)‐C(6) 1.446(8) C(1)‐C(3) 1.528(6) C(1)‐C(2) 1.551(5) C(4)‐C(5) 1.469(8) C(6)‐C(7) 1.264(10) C(8)‐C(9) 1.410(5) C(8)‐C(13) 1.412(5) C(9)‐C(10) 1.376(5) C(10)‐C(11) 1.380(6) C(11)‐C(12) 1.388(5) C(12)‐C(13) 1.367(5) O(1)‐P(1)‐O(2) 115.6(2) O(1)‐P(1)‐O(3) 116.1(2) O(2)‐P(1)‐O(3) 104.2(2) O(1)‐P(1)‐C(1) 113.99(18) O(2)‐P(1)‐C(1) 105.79(17) O(3)‐P(1)‐C(1) 99.4(2) C(8)‐N(1)‐C(1) 130.1(3) O(5)‐N(2)‐O(4) 122.5(3) 49 Table 2.6 Continued O(5)‐N(2)‐C(11) 119.2(4) O(4)‐N(2)‐C(11) 118.3(4) C(4)‐O(2)‐P(1) 125.4(3) C(6)‐O(3)‐P(1) 123.7(6) N(1)‐C(1)‐C(3) 113.7(3) N(1)‐C(1)‐C(2) 105.1(3) C(3)‐C(1)‐C(2) 108.6(4) N(1)‐C(1)‐P(1) 110.5(3) C(3)‐C(1)‐P(1) 111.3(3) C(2)‐C(1)‐P(1) 107.1(3) F(1)‐C(3)‐C(1) 109.9(4) O(2)‐C(4)‐C(5) 110.8(5) C(7)‐C(6)‐O(3) 118.9(7) N(1)‐C(8)‐C(9) 125.6(3) N(1)‐C(8)‐C(13) 116.7(3) C(9)‐C(8)‐C(13) 117.7(3) C(10)‐C(9)‐C(8) 120.4(4) C(9)‐C(10)‐C(11) 120.4(4) C(10)‐C(11)‐C(12) 120.5(4) C(10)‐C(11)‐N(2) 119.8(4) C(12)‐C(11)‐N(2) 119.7(4) C(13)‐C(12)‐C(11) 119.6(4) C(12)‐C(13)‐C(8) 121.3(4) _____________________________________________________________ Symmetry transformations used to generate equivalent atoms: 50 Table 2.7 Anisotropic displacement Parameters (Å 2 x 10 3 ) for C 13 H 17 FN 2 O 5 P. The Anisotropicdisplacement Factor Exponent Takes the Form: 2π 2 [ h2 a*2U11 + ... + 2 h ______________________________________________________________________________________ U 11 U 22 U 33 U 23 U 13 U 12 _____________________________________________________________________________________ P(1) 43(1) 76(1) 44(1) ‐22(1) 15(1) ‐14(1) F(1) 106(3) 122(3) 119(3) ‐22(2) 1(2) 23(2) N(1) 42(2) 52(2) 41(2) ‐11(1) 14(1) ‐8(1) N(2) 49(2) 59(2) 47(2) 13(2) 8(2) ‐4(2) O(1) 49(2) 97(2) 53(2) ‐26(2) 7(1) 2(2) O(2) 54(2) 110(3) 37(2) ‐8(2) 12(1) 14(2) O(3) 75(2) 108(3) 94(3) ‐34(2) 18(2) ‐49(2) O(4) 71(2) 80(2) 53(2) 5(2) 27(2) ‐13(2) O(5) 67(2) 68(2) 61(2) 9(2) 10(2) ‐26(2) C(1) 49(2) 50(2) 41(2) ‐6(2) 16(2) ‐10(2) C(2) 64(3) 68(3) 49(2) 3(2) 18(2) ‐15(2) C(3) 62(3) 53(2) 58(3) ‐8(2) 15(2) ‐7(2) C(4) 68(3) 88(4) 63(3) 7(3) 22(3) ‐2(3) C(5) 144(6) 96(4) 44(3) ‐3(3) ‐9(3) 15(4) C(6) 161(7) 296(13) 67(4) ‐18(6) 10(4) ‐181(9) C(7) 60(4) 293(13) 102(6) ‐55(7) 6(4) ‐35(6) C(8) 39(2) 44(2) 37(2) ‐1(2) 7(1) 0(2) C(9) 45(2) 46(2) 40(2) ‐8(2) 12(2) ‐3(2) C(10) 49(2) 52(2) 39(2) 0(2) 10(2) 4(2) C(11) 40(2) 48(2) 41(2) 8(2) 7(2) 0(2) C(12) 47(2) 47(2) 45(2) 1(2) 3(2) ‐9(2) C(13) 46(2) 49(2) 38(2) ‐6(2) 9(2) ‐3(2) ______________________________________________________________________________________ 51 Table 2.8 Hydrogen coordinates ( x 10 4 ) and Isotropic displacement parameters (Å 2 x 10 3 ) for C 13 H 17 FN 2 O 5 P. ____________________________________________________________________________________ x y z U(eq) ____________________________________________________________________________________ H(2A) 10230 1658 ‐452 90 H(2B) 10103 3296 ‐344 90 H(2C) 8722 2445 ‐697 90 H(3A) 7673 3322 1122 69 H(3B) 8635 4286 615 69 H(4A) 10812 314 2257 87 H(4B) 9445 913 2686 87 H(5A) 12368 1979 2751 143 H(5B) 11565 1227 3399 143 H(5C) 11010 2720 3124 143 H(6) 12747 4079 1831 209 H(7A) 13868 3790 470 182 H(7B) 14802 4276 1218 182 H(9) 7283 1658 1701 52 H(10) 5438 640 2359 56 H(12) 4679 ‐2198 769 56 H(13) 6516 ‐1210 106 53 ________________________________________________________________________________ 52 2.7 Chapter 2 NMR Spectra 19 FNMR of Diethyl1,1difluoro2(4nitrophenylamino)propan2 ylphosphonate 53 31 PNMR of Diethyl1,1difluoro2(4nitrophenylamino)propan2 ylphosphonate 54 1 HNMR of Diethyl1,1difluoro2(4nitrophenylamino)propan2 ylphosphonate 55 13 CNMR of Diethyl 1fluoro2(ptolylamino)propan2ylphosphonate 56 1 HNMR of Diethyl 1fluoro2(ptolylamino)propan2ylphosphonate 57 31 PNMR of Diethyl 1fluoro2(ptolylamino)propan2ylphosphonate 58 13 C NMR of Diethyl 1fluoro2(2fluorophenylamino)propan2ylphosphonate 59 19 F NMR of Diethyl 1fluoro2(2fluorophenylamino)propan2ylphosphonate 60 1 H NMR of Diethyl 1fluoro2(2fluorophenylamino)propan2ylphosphonate 61 31 P NMR of Diethyl 1fluoro2(2fluorophenylamino)propan2ylphosphonate 62 13 C NMR of Diethyl 1fluoro2(phenylamino)propan2ylphosphonate 63 1 H NMR of Diethyl 1fluoro2(phenylamino)propan2ylphosphonate 64 31 P NMR of Diethyl 1fluoro2(phenylamino)propan2ylphosphonate 65 19 F NMR of Diethyl 1fluoro2(phenylamino)propan2ylphosphonate 66 2. 8 Chapter 2 References 1. Allen, M. C.; Fuhrer, W.; Tuck, B.; Wade, R.; Wood, J. M. J. Med. Chem., 1989, 32, 1652. 2 .Logusch, E. W.; Walker, D. M.; McDonald, J. F.; Leo, G. C.; Grang, J. E. J. Org. Chem., 1988, 53, 4069. 3. Giannousis, P. P.; Bartlett, P. A. J. Med. Chem., 1987, 30, 1603. 4. Hirschmann, E.R.; Smith III ,A. B.; Taylor, C. M.; Benkovic, P. A.; Taylor, S. D.; Yager, K. M.; Sprengler, P. A.; Benkovics, S. J. Science, 1994, 265, 234. 5. Hoagland, R. E. Naturally Occurring CarbonPhosphorus Compounds as Herbicides. ACS Sym. Ser. 1988, 380, 182. 6. Emsle, J.; Hall, E. D. The Chemistry of Phosphorus; Harper and Row:London, 1976. 7. Danila, C. D.; Wang, X.; Hubble, H.; Antipin, I. S.; Pinkhassik, E. Bioorg. Med. Chem. Lett., 2008, 18, 2320. 8. Alonso,E.; Solis, A.; del Pozo C., Synlett., 2000, 698. 9. Lejczak, B.; Kafarski, P.; Sztajer, H.; Mastalerz, P. J. Med. Chem., 1986, 29, 2212. 10. Kukhar, V. P.; Solodenko, N. M.; Solodenko, V. A. Biological activity of the phosphorus analogs of aminoacids. Biokhim. Zh., 1988, 60, 95. 11. Quin, L. D. Organophosphorus Chemistry; Wiley‐Interscience: New York, 2000 12. Bégue, J‐P.; Bonnet‐Deplon, D. J. Fluorine Chem., 2006, 127, 992. 13. Biomedical Aspects of Organofluorine Chemistry (Eds.: R. Filler , Y. Kobayashi); Kodansha and Elsevier Biomedical: Amsterdam, 1983. 14. Cherkasov, R. A.; Galkin, V. I. Russian Chem. Rev., 1998, 67, 882. 15. Fields, E. K. J. Am. Chem. Soc., 1952, 74 (6), 1528. 16 Heydari, A.; Karimian A.; Ipaktschi, J. Tetrahedron Lett., 1998, 39, 6729. 17 Azizi , N.; Saidi, M.R. Eur. J. Org. Chem., 2003, 4630. 67 18. Chandrasekhar,S.; Prakash, S.J.; Jagadeshwar V.; a Narsihmulu, Ch.Tetrahedron Lett., 2001, 42, 5561. 19. Ranu, B.C.; Hajra,A.; Jana, U. Org. Lett., 1999, 1, 1141. 20 Manabe, K.; Kobayashi,S. J. Chem. Soc., Chem. Commun., (2000), p. 669. 21 Kaboudin, B.; Rahmani, A. Synthesis, 2003, 2705. 22 Lee, S.; Park, J.H.; Kang, J.; Lee, J.K. J. Chem. Soc., Chem. Commun., 2001, 1698. 23 Akiyama,T.; Sanada, M.; Fuchibe, K. Synlett, 2003, 1463. 24 Bhagat, S.; Chakraborty, A.K. J. Org. Chem., 2007, 72, 1263. 25 Reddy, Y.T.; Reddy, P.N.; Kumar, B.S.; Rajput, P.; Sreenivasulu, N.; Rajitha, Phosphorous, Sulphur, Silicon Relat. Elem., 2007, 182, 161. 26 Heydari, A.; Arefi, A. Catal. Commun., 2007, 8, 1023. 27 Gosh, R.; Maiti, S.; Chakraborty A.; Maiti, D.K. J. Mol. Catal. A: Chem., 2004, 210, 53. 28. Yager, K.M.; Taylor, C.M.; and A.B. Smith, J. Am. Chem. Soc., 1994, 116, 9377. 29. Simoni, D.; Invidiata, F.P.; Manferdini, M.; Lampronti, I.; Rondanin,R.; Roberti, M.; Pollini, G.P. Tetrahedron Lett., 1998, 39, 7615. 30. Klepacz, A.; Zwierzak, A. Tetrahedron Lett., 2002, 43, 1079. 31. Manjula, A.; Rao, V.; Neelakanthan, P. Synth. Commun. 2003, 33, 2963. 32. Doye, S. Synlett., 2004, 1653. 33. Schlemminger, I.; Willecke, A.; Maison, W.; Koch, R.; Lutzen, A.; Martens, J. J. Chem. Soc., Perkin Trans., 2001, 1, 2804. 34. Laschat S.; Kunz, H. Synthesis, 1992, 90 35. Matveeva E.D.; Zefirov, N.S. Russ. J. Org. Chem., 2006, 42, 1237. 68 36. Yadav, J.S.; Reddy, B.V.S.; Raj, K.S.; Reddy B.; Prasad, A.R. Synthesis, 2001, p. 2277. 37. Prakash, G. K. S.; Mathew T.; Panja. C.; Alconcel, S.; Vaghoo.H.; Do, C. Proc. Natl. Acad. Sci. USA, 2007, 104, 3703 38. Olah, G. A.; Farooq, O.; Farnia, S. M. F.; Olah, J. A. J. Am. Chem. Soc., 1998, 110, 2560. 39. Olah, G. A.; Farooq, O.; Cheng, L. X.; Farnia, M. A. F.; Aklonis, J. J. J. Appl. Polym. Sci., 1992, 45, 1355. 40. Prakash, G. K. S.; Yan, P.; Török, B.; Busci, I.; Tanaka, M.; Olah, G. A. Catal. Lett., 2003, 85, 1. 41. Prakash, G. K. S.; Yan, P.; Torok, B.; Olah, G. A. Catal. Lett., 2003, 87, 109. 42. Yan, P.; Batamack, P.; Prakash, G. K. S.; Olah, G. A. Catal. Lett., 2005, 101, 141. 43. Yan, P.; Batamack, P.; Prakash, G. K. S.; Olah, G. A. Catal. Lett., 2005, 103, 165. 44. Deng, X‐M.; Sun, X‐L.; Tang, Y. J. Org. Chem., 2005, 70, 6537. 45. Lacey, J. R.; Anzalone, P. W.; Duncan, C. M.; Hackert, M. J.; Mohan, R. S. Tetrahedron Lett., 2005, 46, 8507. 46. Lacey, J. R.; Anzalone, P. W.; Duncan, C. M.; Hackert, M. J.; Mohan, R. S. Tetrahedron Lett., 2005, 46, 8507. 47. Yan, P. PhD. Thesis, University of Southern California, 2004. 69 Chapter 3. Modified Nucleotide Triphosphate Analogs 3.1 Introduction Deoxyribonucleic acid (DNA) is the building block of all living organisms and is responsible for storing genetic information pertaining to the development and function of all living organisms and viruses. DNA consists of two long polymers of simple units called nucleotides. The nucleotide consists of three structural regions: triphosphate moiety, deoxyribose sugar, and the aromatic base (Figure 3.1). The triphosphate is connected to the C5 ′ carbon of the deoxyribose sugar and the base is connected to the C1 ′ carbon of the deoxyribose sugar molecule via a β‐glycosidic bond. Structurally the triphosphate moiety and the deoxyribose sugar are common in all nucleotides. The variation in the structure of nucleotides is found in the base connected to the C1’of the deoxyribose (Figure 3.1) Figure 3.1 Deoxyribose Nucleic Acid and Purine and Pyrimidine Bases. 70 DNA conserves its genomic integrity through pairing of purine and pyridine bases via hydrogen bonding in the two anti‐parallel strands of DNA. Adenine base pairs by two hydrogen bonds with thymine, while guanine binds with cytosine through three hydrogen bonds (Figure 3.2). These hydrogen bonds are also known as Watson‐Crick base pairing. . Figure 3.2 Two strands of DNA Showing WatsonCrick Base Pairings. DNA polymerase plays a central role in replication and repair in DNA synthesis. DNA polymerase catalyzes the nucleophilic attack of the 3 ′OH end of the existing DNA chain, on the α‐phosphate of an incoming deoxyribonucleotide triposphophate (dNTP) to form a phosphodiester bond, releasing pyrophosphate (PPi) in the process. The energy for this process is driven by the subsequent hydrolysis of pyrophosphate released from the incoming dNTP. As mentioned in 71 Chapter 1, hydrolysis of phosphonate esters releases around ‐7.4 kcal/mol of energy per P‐O bond hydrolysis. To understand the detailed mechanism of the DNA polymerase reaction, it is necessary to obtain substrates with various functional modifications that will test the integrity of natural dNTP structure. 1 The established dogma for high fidelities of DNA polymerases has been attributed to Watson‐Crick base pairing but recently there have been numerous studies related to the base selectivity of DNA polymerase where the bases, purines and pyridines have been modified to address the electronic and structural specificity of the incoming dNTP. Figure 3.3 Modified Nucleotide of Thymidine Derivatives. However Kool et. al. have recently shown that 2,4‐difluorotoluene and other isosteric analogues of thymine (Figure 3.3) 2 can incorporate dNTP in the absence of Watson‐Crick base parings. In another example, Bergstorm et al. developed azole (3.1) and triazole‐carboxamide nucleotides, 3 which allowed universal base paring with purine and pyridine bases by maximizing base stacking interaction (Figure 72 3.4). These studies indicated the preference of geometrical structural conformities rather than hydrogen bonding at the active site of the DNA in determining base pairings. O OH O P O O - O N O 2 N P O P - O O - O - O O 3.1 Figure 3.4 Nucleotide which Allows Universal Base. 3.2 Result and Discussion The active site of various strands of DNA polymerase shows two magnesium ions interacting with primer terminus and the incoming dNTP (Figure 3.5) as suggested by Thomas Steitz. 4 One metal polarizes the hydroxyl group at the 3 ′ primer terminus, facilitating nucleophilic attack by the hydroxyl group at the P α of the incoming dNTP. Both metals are involved in stabilizing a five‐coordinate transition state, where α‐phosphate is linked to five oxygen atoms and the second metal facilitates the eliminating pyrophosphate. Theoretical calculations of the step wise reaction mechanism of DNA polymerase 5 show the binding of dNTP with the polymerase active site combined with the base pairing with the template DNA strand, and is followed by nucleophilic attack of the growing DNA primer at the 3 ′ oxygen (activated by deprotonation) on the P α of the triphosphate moiety with subsequent elimination of pyrophosphate. In 73 the absence of any significant steric difference from modified leaving group, nucleotide incorporation reaction rate is in good correlates correlations with the acidity of the leaving groups as the negative charge builds up at the P α , when the addition step of the primer 3 ′ oxygen is the rate determining step. Thus, the rate of the DNA polymerase reaction is sensitive to the polarization ability of the leaving groups but the effect of polarization will be greater when the elimination of the leaving group is the rate determining step. Figure 3.5 Transition State of the Active Site of DNA Polymerase Reactions. To understand the detailed step wise mechanism of DNA polymerase reaction, various modifications have been envisioned at different positions of the 74 triphosphate linkage. In particular, leaving group properties were evaluated recently in conjecture with theoretical studies. 6,7,8 In these studies various modified triphosphate analogs of dNTP were used to understand the polymerase‐substrate ternary complex, the catalytic efficiency, and the fidelity of human DNA polymerase. Pol β has been used extensively as a model system to investigate fidelity 9,10 due to its crucial role in DNA repair 11 and the availability of numerous crystal structures of various ligand states. 12,13,14 Modification of the triphosphate group can allow the enzymatic reaction of DNA polymerase to be tuned systematically by altering the leaving group properties of the modified dNTP. In one study, single point crystal structure of the ternary complex of the native Pol β with methylene analog of dNTP in the triphosphosphate region in the solvent accessible region, where the β‐γ oxygen is replaced by P‐CF 2 ‐P and CH 2 , showed minimum perturbation when superimposed with crystal structure of the ternary complex of Pol β with natural dNTP. 6 There were also no protein interactions between the bridged β‐γ oxygen or with the β, γ phosphorus from the Pol β. This study also showed that there were no significant energy differences in the position of the catalytic metal cations or the aspartate at the active site of the enzyme, due to these modifications. In this project our goal was to synthesize triphosphate analogs of natural dNTP by initially synthesizing bisphosphonate analogs and then coupling them with nucleotide monophosphate moiety. To begin our studies, we wanted to compare the structural and electronics properties of pyrophosphate unit with known PPi 75 analogues which have previously been synthesized for enzymatic studies. 15,16,17,18,19,20,21,22,23 By using substituent at different positions, the fundamental parameters such as pKa and their binding affinity to metals can be greatly altered, which can affect the course of enzymatic reactions. 3.2.1 Bisphophonic Acid Analogs The CF 2 ‐substituted phosphonic acid derivatives are of of great interest, because CF 2 group is isoelectronic and isosteric to the oxygen atom in PPi. The pK a values of 1.44, 2.11, 5.66, and 7.63 24 of CF 2 based bisphosphonic acid analog is also comparable to that of 0.85, 1.49, 5.77, and 8.22 of PPi. 25 To synthesize CF 2 analogs of pyrophosphonate, the corresponding tetraethyl diflurobisphosphonate (3.3) was synthesized in 70‐75% yields using the known procedure developed by Shipitshin et. al. 26 The tetraethyl difluoromethylenebisphosphonate is then hydrolyzed using the procedure of Mckenna and Shen 27 (Scheme 3.1) to give difluromethylene phosphonic acid (3.4). Scheme 3.1 Synthesis of Difluromethyelenebisphosphonate. 76 The crystal structure of the difluromethylenebisphonic acid was obtained as a m‐toludinium salt and the crystal structure was determined. The crystallographic data and some selected structural parameters for the H[CF 2 PP] 3‐ is shown in Table 3.1 and Table 3.2. 28 Table 3.1 Crystal Data and Structure Refinement for Difluromethylenebisphosphonate, mTuolidium Salt Empirical formula C 44 H 60 F 4 N 6 O 12 P 4 fw 1064.86 cryst color, habit crystal dimens (mm3) 0.25 × 0.20 × 0.10 temp (K) 153(2) wavelength (Å) 0.71073 cryst syst triclinic space group P Formula unit/unit cell 1 a (Å) 12.7629(7) b (Å) 13.3992(7) c (Å) 17.1002(9) α (°) 69.350(2) β (°) 72.000(2) γ (°) 89.762(3) V (Å 3 ) 2584.4(2) Z 2 Density (calcd) (Mg/m 3 ) 1.370 abs coeff (mm ‐1 ) 0.224 F(000) 1118 2θ range (°) 1.35 ≤ 2θ ≤ 27.10 range of h, k, l −16 → 13, −16 → 17, −22 → 13 reflns collected 15 683 independent reflns 10 969 [R(int) = 0.0373] completeness to θ = 27.10° 96.2% abs correction none refinement method full‐matrix least‐squares on F 2 data/restraints/params 10 969/0/647 GOF on F 2 1.006 final R indices [I > 2σ(I)] R1 = 0.0484, wR2 = 0.0879 R indices (all data) R1 = 0.0805, wR2 = 0.0932 largest diff. peak and hole 0.480 and ‐0.346 e.Å ‐3 77 When comparing the structure and electronics of diflurobisphosphonic acid with that of pyrophosphates, the anionic radius of diflurobisphosphonic acid [CF 2 PP] 4 is much larger than that of PP i anions because the bond length of P‐C is much longer than that of the corresponding P‐O (1.597 Å ). 29 The F atoms in the CF 2 unit are occupying the position of the lone pairs of oxygen atom in the PP i unit and contribute to the size enhancement of the [CF 2 PP] 4‐ unit compared to the PP i . The shape of the anion is slightly different since the <P‐O‐P bite angle is different from that of the <P‐C‐P bite angle. Table 3.2 Selected Bond Lengths (Å) and Angles (deg) for C 44 H 60 F 4 N 6 O 12 P 4 P(1)‐O(1) 1.5052(15) P(2)‐O(6) 1.4995(17) P(1)‐O(3) 1.5156(17) P(2)‐O(5) 1.5641(19) P(1)‐O(2) 1.5209(15) P(2)‐C(1) 1.849(2) P(1)‐C(1) 1.866(3) F(1)‐C(1) 1.382(2) P(2)‐O(4) 1.4965(17) F(2)‐C(1) 1.396(2) O(1)‐P(1)‐O(3) 113.87(10) O(4)‐P(2)‐C(1) 104.78(10) O(1)‐P(1)‐O(2) 113.47(09) O(6)‐P(2)‐C(1) 108.34(10) O(3)‐P(1)‐O(2) 111.83(09) O(5)‐P(2)‐C(1) 103.60(10) O(1)‐P(1)‐C(1) 107.18(10) F(1)‐C(1)‐F(2) 104.61(17) O(3)‐P(1)‐C(1) 106.05(10) F(1)‐C(1)‐P(2) 107.13(15) O(2)‐P(1)‐C(1) 103.49(10) F(2)‐C(1)‐P(2) 108.67(15) O(4)‐P(2)‐O(6) 116.59(11) F(1)‐C(1)‐P(1) 108.26(15) O(4)‐P(2)‐O(5) 109.80(11) F(2)‐C(1)‐P(1) 107.88(15) O(6)-P(2)-O(5) 112.60(10) P(2)-C(1)-P(1) 119.34(12 78 There were no unusual structural parameters on the crystal structure of the m‐toludinium counterion. The molecules packs as a dimer with one molecule of H[(O 3 P) 2 CF 2 ] 3‐ and one molecule [(O 3 P) 2 CF 2 ] 4‐ is connected through hydrogen bonding (Figures 3.6). The dimeric units combine to make a super structure with sheets of anions linked by H‐bonding between units. The presence of hydrogen bonding was supported by calculation using an appropriate HFIX command, which showed some electron density around oxygen atom and assigned hydrogen to it. Figure 3.6 shows H‐bonding between H100 from one anion and O8 from another. The O8‐H100 distance was found to be 1.64(3) Å, which is well within the distances commonly observed in hydrogen bonds. Figure 3.6 Two Molecules of H[(O 3 P) 2 CF 2 ] 3 Interconnected Through HBond. 79 The major difference between the [(O 3 P) 2 CF 2 ] 4‐ anion and the PPi anion is in the P‐C and P‐O bond length and the bridging angles. The calculated P‐O bond lengths of PP i are 1.68 Å, whereas the calculated P‐C bonds of CF 2 PP are 1.87 Å. The calculated bridging angle (<P‐O‐P) of PPi is 127.6° and the calculated bridging angle (<P‐C‐P) of CF 2 PP is 122.9°. The CF 2 PP unit is therefore longer and bulkier than PP i . Under biological conditions, PP i generally coordinates in a bidentate manner to metal ions such as magnesium 30,31 even though the anion can coordinate in a monodentate fashion. 29 The CFH analog of pyrophosphate was synthesized from the commercially available tertraisopropyl methylenebisphosphonate in 55% isolated yield, using Selectoflur ® as shown in Scheme 3.2, to obtain tetraisopropyl fluromethylenebisphosphonate (3.7) Scheme 3.2, which was then hydrolyzed to the corresponding acids (3.9). The methylene bisphosphonic acid (3.10) was synthesized from the commercially available tertraisopropyl methylene bisphosphonates by hydrolysis in almost quantitative yield. Fluromethylene bisphosphonic acid (3.9) and methylene bisphosphonic acid (3.10) both produced amorphous solids when complexed with m‐toludine and various other bulky nitrogen bases. Hence, we were not able to evaluate the crystal structure of these analogs. 80 Scheme 3.2 Synthesis of MonoFlurobisphosphonate. 3.2.2 Triphosphoric Acid Analogs Triphosphates are of great of interest in the biological system as a part of dNTP. Our goal was to create triphosphate analogs where the oxygen atoms between the three phosphorus atoms are replaced by non hydrolysable analogs, CH 2 , CHF, or CF 2 (Figure 3.7) . The symmetric X=Y=CH 2 triphosphonic acids have previously been synthesized, 32 but the asymmetric CF 2 and CH 2 or the CFH and CF 2 have yet to be synthesized. Figure 3.7 Analogs of Triphosphonic Acids. 81 We initially wanted to synthesize the triphosphosphate acid, where X=Y=CF 2, starting with the synthesis of the corresponding ester (3.12) using the route shown in Scheme 3.3. However, it is interestering to note that the lithiated anion 3.11 did not react with dichloroethylphosphosphonate but rather scrambled into undetermined products. It was observed that the base used in generating the lithiated anion 3.11 is important in determining nucleophilicity of ‐ CF 2 anion. The CF 2 analog of triphosphosphonic acid where X=Y=CF 2 was synthesized by a different route. 33 Scheme 3.3 Synthesis Ethylester of CF 2 Analog of Triphosphate. In another attempted, we tried to prepare symmetric CH 2 analogs of triphosphonate esters (X=Y=CH 2 ) , using a similar procedure as shown in Scheme 3.3, using diethyl methylphosphonate (3.13). Surprisingly, instead of producing the triphosphonate ethyl ester, ethoxy(methyl)phosphoryl)methylphosphonate (3.14) 34 was produced in 59% yield. 82 Scheme 3.4 Synthesis of Fluorinated Ethoxy(methyl)phosphoryl)methylphosphonate. With ethoxy(methyl)phosphoryl)methylphosphonate (3.14) in hand, we tried to synthesize the fluorinated deriviatves 3.15 and 3.16 (Scheme 3.4) with Selectfluor ® . The hydrogen of the methylene group between the phosphorus atoms of 3.14 is relatively more acidic than the terminal methyl and should be easily deprotonated with standard base, such as NaH and tButO ‐ K + , as was done in fluormethylenebisphonate synthesis. When the deprotonated species was subjected to electrophilic fluorination using Selectfluor ® or NSF. (Scheme 3.4), fluorination did not take place, but starting 3.14 compound was recovered after work up. 3.2.3 Monophonic Acid Analog of Bisphophonic Acid The active site of DNA polymerase shows no interaction with the terminal γ phosphorus atom of the incoming dNTP as mentioned previously. This opened up a whole new area of structural modification that can take can be affected at the terminal γ phosphorus atom. We envisioned that replacing the γ phosphorus atom with other tetrahedral structures can increase the structural variation in modified nucleotide analogs. 3,3,3‐trifluoro‐2,2‐dihydroxypropylphosphonic acid (3.19) was synthesized (Scheme 3.5) specifically to address the structural variation in 83 bisphosphonate with dihydroxy carbonyl group –[C(OH) 2 ]‐. The diethyl methylphosphonate (3.13) was reacted with ethyl 2,2,2‐trifluoroacetate to produce diethyl 3,3,3‐trifluoro‐2,2‐dihydroxypropylphosphonate (3.17). Diethyl 3,3,3‐ trifluoro‐2‐oxopropylphosphonate and diethyl 3,3,3‐trifluoro‐2‐hydroxyprop‐1‐ enylphosphonate were also produced, but during acidic work up, both compounds converted to 3.17 in 65% yield. The ester 3.17 is a crystalline solid hence was easily separated from the unreacted starting materials. The ester 3.17 is subsequently hydrolyzed to provide quantitative yield of the acid 3.19, as a colorless solid. Scheme 3.5 Synthesis of 3,3,3Trifluoro2,2dihydroxypropylphosphonic Acid Since the pKa of bisphosphonic acids are very important, we also evaluated the pKa of 3,3,3‐trifluoro‐2,2‐dihydroxypropylphosphonic acid (3.19) by titrating with 0.05 M of NaOH solution. The first pKa of 3,3,3‐trifluoro‐2,2‐ 84 dihydroxypropylphosphonic acid was found to be 4.43 and the second pKa was found to be 8.21, which is close to the third and fourth pKa values of both pyrophosphate and difluromethylenebisphosphonic acid. 18,19 Figure 3.9 shows the titration curve for determining the pKa value of 3,3,3‐trifluoro‐2,2‐ dihydroxypropylphosphonic acid. Figure 3.8 Tritration Graph 3,3,3trifluoro2,2dihydroxypropylphosphonic Acid with 0.05 M NaOH Solution. 3.2.4 Modified Nucleotide Analog The synthesis of nucleotide triphosphate analogs was carried out as shown in Scheme 3.6. The morpholidate derivative was synthesized using the know procedure of Moffat et. al 35,36 in 75% yield. The nucleotide analog 3.21 was 0 2 4 6 8 10 12 0 5 10 15 20 25 30 35 40 45 50 Volume of 0.05M NaOH solution pH 85 synthesized by dissolving 3.19 in pyridine at room temperature reacted with 3.20 and stirring the mixture for 3 days. The 31 P NMR showed two doublets at 10.21ppm and ‐10.32 ppm corresponding to P α and P β respectively after removal of pyridine from the reaction mixture. These values are consistent with the 31 P NMR of P α and P β of other 5 ′deoxyribose thymidine triphosphate analogs. 6,7,18 The reaction mixture was passed through the Dowex‐NH 4 + column, which gave better resolution in the 31 P‐NMR spectrum. The product was separated using DEAE‐5PW HPLC column with gradient using 1M NaCl/H 2 O. The product was also separated with an anion exchange column using DEAE Sephadex ® resin with 0.3 M NaCl/H 2 O solution. The nucleotide analog (3.21) slowly decomposed when water was removed by lypholization. When the product was collected and subject to HPLC ion exchange chromatography, it completely decomposed to the starting materials. Scheme 3.6 Synthesis Nucleotide Triphosphate Analogs. We subjected the crude reaction mixture to various pH, ranging from 4 to 7 in hope of separating the product without decomposition. In its crude reaction mixtures 3.21 was found to be stable in pH ranging from 4 to 7 over several days, 86 but upon isolation on HPLC column, it decomposed to the corresponding nucleotide monophosphate and the 3,3,3‐trifluoro‐2,2‐dihydroxypropylphosphonic acid. 3.3 Conclusion In this project various pyrophosphonic acid analogs of bisphosphonic acids and 3,3,3‐trifluoro‐2,2‐dihydroxypropylphosphonic acid were synthesized to couple with nucleosides to generate both hydrolysable and non‐hydrolysable nucleotide analogs. These nucleotides analogs are crucial for further studies on fidelity mechanism of DNA polymerase and to develop insight into developing drugs for the treatment of HIV and cancer, where the DNA polymerase plays significant role in the course of the disease. 3.4 Experimental 3.4.1 General Unless otherwise mentioned, all chemicals were purchased from commercial sources. THF was dried over sodium under nitrogen 1 H, 13 C, 19 F, and 31 P NMR spectra were recorded on Varian Mercury series NMR spectrometers at 400 MHz. 1 H NMR chemical shifts were determined relative to internal tetramethylsilane at δ 0.0 or the 1 H signal of CDCl 3 unless otherwise stated. 13 C NMR chemical shifts were determined relative to internal tetramethylsilane at δ 0.0 or to the 13 C signal of CDCl 3 at δ 77.0. 19 F NMR chemical shifts were determined relative to external standard CFCl 3 at δ 0.0 31 P NMR chemical shifts were determined with external 87 standard H 3 PO 4 at δ 0.0 GCMS data were obtained from the Thermo‐Finnigan DSQ GC‐mass spectrometer. Ion exchange chromatograph with DOWEX 50‐200 mesh as well in HPLC using DEAE‐5PW HPLC column. 3.4.2 Synthesis Relevance to Chapter 3 Synthesis of Diethyl Difluoromethylphosphonate(3.2) Diethyl difluromethlyphosphate (3.2) was synthesized from the known procedure published by the work of Burton et al. 37 In a three neck 250 mL round bottom flask, equipped with condenser, was charged with 100 mL of dry THF and 25.825 g (0.187mol) of diethyl phosphate was taken. To this, 4.5 g (0.196mol) of sodium was carefully added slowly and stirred until it completely dissolved. The condenser was replaced with dry ice/acetone condenser and CF 2 ClH (0.196mol) was bubbled through for 2 hrs. The solution turned murky and gelatinous after which the reaction mixture was stirred over night and then quenched with 50 mL of water. The white gelatinous solid was dissolved and the product was extracted three times with 50 mL diethyl ether in brine solution. The organic layer was collected, dried over MgSO 4 , filtered and the solvent removed to form a yellowish liquid. The yellowish liquid was transfered into 100 mL round bottom flasked and vacuum distilled to give a 23.12g of clear liquid in 66% yield. 88 Synthesis of Tetraethyl Difluoromethylenediphosphonate (3.3) The synthesis of tetraethyl difluoromethylenediphosphonate (3.3) was based on a modified procedure by Shipitshin et al. 26 In a typical preparation, a 100 mL Schlenk flask equipped with a magnetic stirring bar and rubber septum was charged under dry nitrogen with THF (10 mL, freshly distilled from sodium benzophenone ketyl) and i‐Pr 2 NH (1.80 mL, 12.8 mmol, freshly distilled from NaOH) and placed in an ice‐water bath. After 30 min of stirring, a 2.45 M n‐BuLi solution in hexanes (5.30 mL, 13.7 mmol) was added drop‐wise within 6 min. After another 30 min, the ice‐ water bath was replaced by a ‐78 °C isopropanol‐dry ice slush bath, and the reaction mixture was stirred for an additional 30 min before a solution of diethyl difluoromethyl phosphonate (2.26 g, 12.0 mmol) in THF (9 mL) was added drop‐ wise within 6 min. Thirty minutes later, a solution of diethyl phosphochloridate (2.05 g, 11.9 mmol) in THF (9 mL) was added dropwise to the brown reaction mixture within 8 min. After 1 h of stirring at ‐78 °C, the isopropanol‐dry ice bath was replaced by an ice water bath and the stirring was continued for another hour before a saturated aqueous solution of KH 2 PO 4 (10 mL) was added to the reaction mixture. After being stirred at ambient temperature overnight, the contents of the flask were transferred into a separatory funnel with the aid of EtOAc (80 mL) and water (40 mL). The aqueous phase was extracted twice with 50 mL portions of EtO P P OEt O O OEt OEt F F 89 EtOAc, and the organic phases combined and dried over MgSO 4 . After filtration and removal of solvent and any volatiles under reduced pressure, the crude product was obtained as yellow oil. It was purified by column chromatography on silica gel (EtOAc/hexanes, 3:2). Additional purification was achieved by heating the product under vacuum (75 °C, 3 mmHg) overnight. The product was a colorless oil, yield 2.71 g (70%). Typical Synthesis of Bisphosphonic Acids and Other Phosphonic Acids From Their Corresponding Esters The syntheses of bisphosphonic and phosphonic acid analogs were achieved according to a modified procedure of McKenna and Shen. 27 In a typical synthesis, a 50 mL round‐bottomed flask (dried under vacuum using a heat gun) containing tetraethyl methylenebisphosphonates (8.0 mmol) and a magnetic stir bar was charged under dry nitrogen with 98% bromotrimethylsilane (7.00 mL, 52.0 mmol). The flask was sealed with a glass stopcock, and the reaction mixture was stirred at ambient temperature for 4.5 days. The volatiles were removed in vacuo followed by addition of water (14 mL). After being stirred for 50 min at ambient temperature, the colorless liquid was transferred into a separator funnel, and the organic phase was separated and discarded. The aqueous phase was washed twice with 15 mL portions of ether and transferred into a 100 mL round bottomed flask with water (combined volume 30 mL). Most of water was removed in vacuum at ambient temperature. The resulting oil was dried over P 2 O 5 in vacuo (0.1 mmHg, 4 days), the 90 wet P 2 O 5 being replaced with fresh dry material at least once. Difluoromethylenebisphosphonic acid as colorless crystals, yield 1.68 g (96%). Spectral Data of 3,3Trifluoro2,2dihydroxypropylphosphonic Acid (3.19) 31 PNMR δ 23.095 (s), 1H‐NMR δ 2.11 (dd, 2H, 3 J F‐H =5.8Hz, 2 J P‐H ‐F=18.7Hz), 19 F‐NMR δ ‐86.95. Preparation of the mToludinium Salt of Difluromethylene Bisphosphonic Acid (3.5) for Crystallographic Analysis. Difluoromethylenebisphosphonic acid (50 mg, 0.33 mmol) was dissolved in 10 mL of deionized water. The solution was treated with excess m‐toluidine (0.500 mL, 4.67 mmol) under stirring. A sticky brown precipitation was formed, which was filtered and recrystallized from deionized water was carried out to obtain white crystals. Xray Crystallography Diffraction data for the H[CF 2 PP] 3‐ anion were collected at 153 K on a SMART APEX CCD diffractometer with graphite‐monochromated Mo K α radiation(ì ) 0.71073 Å). A hemisphere of the crystal data was collected up to a resolution of 0.75 Å. Cell parameters were determined using SMART software. The SAINT package was used for integration of data, Lorentz, polarization, and decay corrections, and for the merging of data. No absorption correction was applied. All calculations for structure determination were carried out using the SHELXTL package (version 5.1). Initial atomic positions were located by direct methods using XS, and the structure was refined by least‐squares methods using SHELX with 10 969 independent reflections 91 and within the range of θ= 1.35‐27.10° (completeness 96.2%). Calculated hydrogen positions were input and refined in a riding manner along with the attached carbons. Hydrogen atoms involved in H‐bonding were located using the appropriate HFIX command. Views of the structure were prepared using ORTEP3 for Windows. Synthesis Tetraisopropyl Fluoromethylenediphosphonate (3.7) In a three neck round bottom flask 0.652 g of NaH was taken with 40 mL of dry THF, under nitrogen atmosphere and cooled to 0 °C. To this 8 g of tetraispropyl methylene diphosphonate in 10 mL of dry THF was added drop wise and reaction mixture brought to room temperature and stirred until effervescence ceased. The reaction vessel was brought back to 0 °C and then 6.215g of Selectrofluor ® in 3 mL of dry DMF was added. The solution turned orange and the reaction mixture stirred for 3‐4 hrs. The reaction mixture was quenched with 30 mL 1M HCl solution and the aqueous layer separated from the organic layer. The aqueous layer was washed with 30 mL of ethyl acetate 3 times and the solvent of the combined organic layer removed under reduced pressure. The products were subjected to column chromatography on silica gel using 1:3 ratio of ethylacetate/hexane as elutant. and Spectral data compared with literature and were found to be the same. 6 92 Synthesis of Diethyl (ethoxy(methyl)phosphoryl)methylphosphonate (3.14). In a 100 mL round bottomed flask, under nitrogen, 4g of diethyl methylene phosphonate was taken in 20 mL of dry THF and the reaction vessel cooled to ‐78 °C in a dry ice‐isopropanol bath. To this 10 mL of nBuLi (2.5M in hexane) was added dropwise and the reaction mixture stirred for 2 hrs at ‐78 °C and then slowly brought up to room temperature and stirred for another 2hrs. The reaction mixture was quenched with 20 mL 3M HCl and the organic layer was separated. The aqueous layer was washed three times with 30 mL of ethyl acetate and then combined organic layer was washed three times with water, then dried over MgSO 4 and the solvent removed. The product was purtified by column chromatography on silica gel with with 1:9 mixture of methanol and methylene chloride to give 2.36g of 3.14 in 59 % yield. Spectral data consistent with the literature. 34 Synthesis of Diethyl 3,3,3trifluoro2,2dihydroxypropylphosphonate (3.19) Typical synthesis of diethyl‐3,3,3‐trifluro‐2,2‐dihydroxyprophylphosphonate involved treating 2g of diethyl methyl phosphonate (13.2mmol) in a 50 mL round bottom flask (dried under vacuum using heating gun), with 2g OEt P F 3 C OH HO O OEt EtO P P CH 3 O O OEt OEt H H 93 diethylmethylphosphonate in 20 mL dry THF, cooled to ‐78 °C using dry ice and acetone slush bath for 10 minutes. 8 mL of 1.6 M n‐butyllithium was added to and the reaction mixture and stirred for one hour. To this, trifluromethyl acetate in 20 mL of dry THF was added drop wise using a syringe and the reaction mixture brought to room temperature. The reaction mixture was quenched with 1M HCl solution. The organic layer separated and the aqueous layer washed twice with 20 mL portions of CH 2 Cl 2 . The organic layer was dried with magnesium sulfate and filtered into 250 mL round bottom flask and the solvent removed to produce a white crystalline solid. Spectral Data for Diethyl 3,3,3trifluoro2,2dihydroxypropylphosphonate (3.17) 1 H‐NMR δ 1.32 (m, 6H), 2.28 (d, 2H, 2 J P‐H =19.3Hz), 4.16 (m, 4H) 13 C‐NMR δ 16.47 (d, 1C, 3 J P‐C =6.5Hz), 29.94 (d, 1C, 1 J P‐C =139.6Hz), 63.10 (d, 1C, 2 J P‐C =6.3Hz) 122.49 (m, 1C) 19 F‐NMR δ ‐87.860. 31 P‐NMR δ 26.681. Titration of 3,3,3Trifluoro2,2dihydroxypropylphosphonic Acid (3.19) with 0.05 M aqueous NaOH solution. 3,3,3‐Trifluoro‐2,2‐dihydroxypropylphosphonic acid (1g) was dissolved in 1 mL of deionized water. 0.1 mL of this solution was taken in a conical flask and the 20 mL of deionized water was added. The solution was titrated with 0.05M NaOH solutions and the pH monitored with a pH meter. Table 3.3 shows the volume vs pH data of the titration. 94 Table 3.3 Titration Data for 3,3,3Trifluoro2,2 dihydroxypropylphosphonic Acid with NaOH Synthesis of 5 ′Monophosphate Adenosine Deoxyribose Morpholidate (3.20) Compound 3.20 was synthesized using modified synthesis of Khorana et. al. 35,36 In a 100 mL three neck round bottom flask equipped with stirring bar, condenser and septum, 500 mg of thymidine monophosphates disodium salt was taken in to 15 mL t‐butyl alcohol and 15 mL of water under nitrogen. To this 649 mg of morpholine was added and the mixture brought to reflux until the white solid 95 dissolved to form almost a clear solution. 1.55g of DCC (N,N'‐ Dicyclohexylcarbodiimide) in 15 mL of t‐butyl alcohol was added slowly over three hours and the resulting solution was kept under reflux for another three hours. The reaction mixture was allowed to cool to room temperature and a cream precipitate was observed to form as the reaction proceeded. The solution was filtered and the residue washed with minimum amount of Methanol. The filtrate was evaporated to dryness and the solid formed was dissolved in minimum amount of methanol. To this mixture 20 mL diethyl ether was added. A creamy precipitate was formed. The solvent was evaporated to dryness to produce the 0.783g of product in 76% yield. 5 ′Adenosine Deoxyribose Nucleotidetriphosphate Analogs (3.21) Morphorlidate derivative of adenosine monophosphates (3.20) (200mg) was taken in 10 mL of dry pyridine in a glove box, and to this 200mg of phosphonic acid derivative (3.19) in 10 mL of dry pyridine was added and the reaction mixture stirred at room temperature for 3 days. The pyridine was removed under vacuum and the product subjected to ion exchange column using DOWEX 50WX8‐200 resin in the ammomium (NH 4 + ) form and eluted with de‐ionized water. The product was obtained as a mixture of starting 3,3‐trifluoro‐2,2‐dihydroxypropylphosphonic (3.19) acid and starting nucleoside (3.20) as well as other unidentified products. Water was removed from the reaction mixture by lypholization. The product was subjected to HPLC DEAE‐5PW column under gradient conditions H 2 O/NaCl (1M) 96 and fractions were collected. Product was obtained at 24 minutes of elution. Water was removed by lypholization and the product analyzed. Spectral Data of 5 ′Adenosine Deoxyribose Nucleotidetriphosphate Analog (3.21) 1 H‐NMR δ 2.29 (dd, 1H, J=6.3Hz, J=19.1Hz), 2.67 (m, 1H), 2.88 (m, 1H), 4.17 (m, 1H), 4.34 (m, 1H), 4.77 (m, 1H), 6.52 (t, 1H, J=6.7Hz), 8.26 (s, 1H) 8.44 (s, 1H). 31 P‐NMR δ ‐10.20 (d, 1P, 2 J P‐P =24.1Hz), 10.14 (d, 1P, 2 J P‐P =23.3Hz). 19 F NMR δ ‐87.286. 97 3.5 Chapter 3 NMR and HPLC Spectra 31 PNMR of 5 ′Adenosine Deoxyribose Nucleotidetriphosphate Analog (3.21) 98 19 FNMR of 5 ′Adenosine Deoxyribose Nucleotidetriphosphate Analog (3.21) 99 1 HNMR of 5 ′Adenosine Deoxyribose Nucleotidetriphosphate Analog(3.21) 100 HPLC Analysis of 5 ′Adenosine Deoxyribose Nucleotidetriphosphate Analog (3.21) 101 19 FNMR of Diethyl (ethoxy(methyl)phosphoryl)methylphosphonate 102 1 HNMR of Diethyl (ethoxy(methyl)phosphoryl)methylphosphonate. 103 13 CNMR of Diethyl (ethoxy(methyl)phosphoryl)methylphosphonate. 104 31 PNMR of Diethyl (ethoxy(methyl)phosphoryl)methylphosphonate. 105 31 PNMR of3,3,3Trifluoro2,2dihydroxypropylphosphonic Acid (3.19) 106 19 FNMR of 3,3,3Trifluoro2,2dihydroxypropylphosphonic Acid (3.19) 107 1 HNMR of 3,3,3Trifluoro2,2dihydroxypropylphosphonic Acid (3.19) 108 3.6 Chapter 3 References 1 .Engel, R. 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Halogenated Trimethylsilane and Nitrate Salt as an Efficient Reagent System for the Direct αHalogenation of Carbonyl Compounds 4.1 Introduction The conversion of C‐H bonds to C‐X bonds (X = F, Cl, Br and I) has a variety of application in medicinal chemistry. The C‐X bonds can diminish metabolic activity as well as increase bioavailabilty. 1 α‐Halogenated carbonyl compounds are important synthetic intermediates and are used as precursors for various organic transformations. 2 α‐Haloacetophenone derivatives have been investigated for their active participation in the inhibition of protein tyrosine phosphatases (SHP‐1 and PTP1B) (Scheme 4.1). 3 α‐Bromoacetophenone derivatives have better inhibition effect than the corresponding chloride derivatives. Therefore by derivatizing the phenyl ring, the properties of PTP inhibitors can be significantly modified. It has been found that the derivatization of the phenyl ring with tripeptide Gly‐Glu‐Glu results in a potent, selective inhibition against a PTP1B. There are relatively few reagents known, that allows direct α‐halogenation of carbonyl compounds. There are significant number of chlorinating reagents are available, which includes trichloroisocyanuric acid, 4 N‐chlorosuccinimide, 5 sulfuryl chloride, 6 polymer supported chlorine 7 and also p‐toluenesulfonyl chloride. 8 Herein, we report α‐chlorination as well as of carbonyl compound with nitryl chloride or bromide generated in situ from chloro/bromotrimethylsilane and nitrate salt, as a source of mild chlorinating and brominating reagent. 112 Scheme 4.1 αBromoacetophenone Derivatives as Potent PTP Inhibitors 4.2 Results and Discussion The chemistry of nitryl chloride (NO 2 Cl) as a reagent has been extensively investigated for nitration of aromatic and aliphatic compounds. 9,10,11,12,13,14,15,16 Nitration typically takes place by electrophilic attack on aromatics, facilitated by Lewis acids. In other studies, nitryl chlorides have been reported to react vigorously with ammonia to generate chloroamine, hydrazine and ammonium nitrite, suggesting that NO 2 Cl can behave as a source of positive chlorine and negative nitrite ions .17,18,19,20,21,22 Scheme 4.2 Generation of NO 2 Cl in situ 113 The convenience of generating nitryl and nitrosyl chloride in situ using chlorotrimethylsilane (TMSCl) and nitrate/nitrite salts was first suggested by Lee et a.l, for deoximination of aldoximes/ketoximes in non‐aqueous medium. 23,24,25 Our group has previously reported 26 the use of ammonium nitrate and chlorotrimethylsilane with a catalytic amount of AlCl 3 as a robust nitrating reagent for the electrophilic nitration of aromatic compounds, which prompted us to develop an in situ nitrating agents using TMSCl/nitrate salts. Recently, we have achieved regioselective nitration of arylboronic acids using chlorotrimethylsilane and nitrate salts. 27 Ipso‐substituted nitro‐aromatic compounds were obtained in high yields and purity in most of these reactions (Scheme 4.2). Scheme 4.3 IpsoNitration of Arylboronic Acids with in situ Generated NO 2 Cl The oxidizing potential of the chlorotrimethylsilane‐nitrate salt reagent system has also been closely examined since most nitrating agents have shown to have oxidizing properties. 28 During our studies of ipso‐nitration of arylboronic acids, we found small amount of nitrochlorination of the arylboronic acids taking place (Scheme 4.3), 27 indicating the possible generation of positive chlorine ion and the oxidizing potential of nitro group in the reaction medium. 114 One of the important oxidation reactions in synthetic organic chemistry is the oxidation of sulfur compounds. Oxidative chlorination of sulfur compounds to sulfonyl chlorides serves as an important step in organic synthesis .29,30,31,32,33,34,35,36,37,38,39,40 Oxidation of sulfur compounds is usually achieved with peroxy compounds such as peroxy acids and hydrogen peroxide. We have previously shown that the direct oxidative chlorination of thiols and disulfides to the corresponding sulfonyl chlorides can be coveniently carried out with chlorotrimethylsilane‐nitrate salt reagent system (Scheme 4.4). 41 The major advantage of this protocol is that in most cases, products obtained need no further purification. Simple removal of the solvent from the reaction mixture provides analytically pure product in most of these cases. Scheme 4.4 TMSClNitrate Salt System as an Efficient Reagent for Oxidative Chlorination of Sulfides and Disulfides During the oxidation of sulfides and sulfoxides, it has been found that the reaction of substrates with acidic α‐H such as methyl phenyl sulfide and sulfoxide yielded a mixture with substantial amounts of chlorine substituted products. This is probably due to the formation of silyl‐enol ether type intermediate (4.3, 4.4) from 115 sulfide and sulfoxide having acidic α‐H (Figure 4.1) which can undergo successive chlorination to give rise to different chlorination products. We found that dialkyl sulfides and sulfoxides also gave a mixture of products and their reactions were also not clean due the competing α‐chlorination. Figure 4.1 Silylenol Ether Type Intermediate from Sulfide and Sulfoxide This prompted us to screen the activity of the chlorotrimethylsilane‐nitrate salt system for α‐chlorination of ketones having α‐H. We have considered acetophenone and its derivatives as convenient substrates. We generated nitryl chloride by reacting TMSCl with potassium nitrate in dichloromethane. Generation of nitryl chloride is indicated by formation of brown gas in the reaction medium which results from decomposition of NO 2 Cl to nitrosyl chloride (NOCl), chlorine gas and nitric acid in the presence of moisture. 21 Acetophenone derivatives are then added and the reaction was carried out at 40 °C for several hours, depending on the substrates. The reaction was monitored by 1 H NMR spectroscopy by taking small aliquot from the reaction mixture. The products are purified by filtration to remove the insoluble nitrate salts, and then subjected to silica gel flash chromatography with hexane. The results are shown in Table 4.1.The chlorination of acetophenone 116 derivatives took place at a relatively slower rate at 40 °C and than at 60 °C, but only 5% dichlorination product was formed. The reaction also took place in the absence of solvent but preceded at a lower rate, and even after 16hrs, significant amount of starting material remained unreacted. Table 4.1 αChlorination of Acetophenones with TMSClNitrate System Ar CH 3 O Ar CH 2 Cl O TMSCl/KNO 3 CH 2 Cl 2 /40 o C 16 16 16 16 16 18 48 43 64 CH 3 O CH 2 Cl O CH 3 O F 3 C CH 3 O Cl CH 3 O H 3 C CH 3 O Cl CH 3 O F CH 3 O CH 3 CH 2 Cl O F 3 C CH 2 Cl O Cl CH 2 Cl O H 3 C CH 2 Cl O Cl CH 2 Cl O CH 3 CH 2 Cl O F Substrate Time (h) Product Yield(%) 63 Cl 42 Cl 73 68 67 * Reactionswerecarriedoutat60 o C,Dichlorinatedproducts7-22% wereobtained 117 A control reaction was carried out where acetophenone was reacted with only TMSCl at 40 °C for 24 hours; no chlorination took place, indicating that chlorination resulted from NO 2 Cl generated in situ when TMSCl was mixed with potassium nitrate salt. ortho‐Substituted acetophenones reacted sluggishly and in some cases the reaction did not go to completion even after subjecting the mixture to 60 °C for 48 hrs. Substrates such as p‐nitroacetophenone, p‐ hydroxyacetophenone, p‐methoxyacetophenone yielded multiple products, which could not be identified. In order to probe the versatility of this reagent system for other halogens, bromotrimethylsilane with potassium nitrate salt was reacted with acetophenone derivatives indicating α‐bromination (Table 4.2). The generation of NO 2 Br was much faster and proceeds at room temperature within a couple of hours of vigorous stirring. This reaction can be accelerated with heat, in which case NO 2 Br formed within few minutes. The α‐bromination was also achieved relatively clean and the product was filtered to remove salts and the solvent was evaporated. 118 Table 4.2 αBromination of Acetophenones With TMSBrNitrate System Taking into account the results of various studies on trimethylsilyl nitrate 26,42 and nitryl halide, 43,44 there are two plausible mechanisms (Scheme 4.5 and Scheme 4.6). This may involve the initial trimethylsilyl nitrate from trimethylsilyl halide and metal nitrate, followed by the formation of nitryl halide (from the interaction of 119 trimethylsilyl nitrate and a second molecule of trimethylsilyl halide), is suggested. In solution, formation of an equilibrium involving trimethylsilyl nitrate and nitryl halide is possible. Similar to the addition of TMSCN, addition of trimethylsilyl nitrate on the carbonyl function (4.5) followed by elimination‐addition step involving nitryl halide (4.6), where the halogen is acting as a nucleophile, which can provide the expected product (Scheme 4.5.). In the alternate mechanism (Scheme 4.6), during the elimination‐addition step, halogen in the nitryl halide can also act as an electrophile, generating the expected product. Formation of nitrogen dinitrogen tetraoxide(N 2 O 4 ) is clearly visible by the brownish yellow color formed during the reaction. Scheme 4.5 Plausible Mechanism for αHalogenation of Acetophenones with Negative Halide Species 120 Scheme 4.6 Plausible Mechanism for αHalogenation of Acetophenones with Positive Halide Species. 4. 3 Conclusions We have reported a simple and mild reagent for α‐chlorination and bromination of ketones with acidic α–hydrogens. α–Bromination and chlorination occurred with good conversion and selectivity. This new method is very simple, mild and very convenient, using less expensive and easily accessible reagents. α– Haloacetophenones with various substituents in the phenyl ring can be prepared under mild conditions and can be scanned for their therapeutic applications as PTP inhibitors. 121 4. 4 Experimental 4.4.1 General Unless otherwise mentioned, all chemicals were purchased from commercial sources. Nitrate salt was dried over P 2 O 5 over night at 60 o C. TMSCl was distilled before use. 1 H, 13 C and 19 F NMR spectra were recorded on a Varian NMR spectrometer at 400 MHz. 1 H NMR chemical shifts were determined relative to internal tetramethylsilane at δ 0.0. 13 C NMR chemical shifts were determined relative to internal tetramethylsilane at δ 0.0 or to the 13 C signal of CDCl 3 at δ 77.0. 19 F NMR chemical shifts were determined relative to internal CFCl 3 at δ 0.0. GCMS data were obtained from Thermo‐Finnigan DSQ GC‐mass spectrometer. 4.4.2 General Procedure for the Halogenation Reaction In a Nalgene ® bottle, to acetophenone (2 mmol) in dichloromethane (10 mL), nitrate salt (4 mmol) and TMSX (X= Cl, Br) (8 mmol) were added. The heterogeneous mixture was stirred vigorously at 40 °C until the reaction went to completion. The reaction mixture was then filtered and solvent removed under reduced pressure. The chlorinated acetophenone derivatives were obtained upon purification using silica gel flash chromatography with hexane. 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Asset Metadata

Creator Ismail, Rehana (author)

Core Title Bisphosphonates for modified nucleotide synthesis and related chemistry

Contributor Electronically uploaded by the author (provenance)

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 07/29/2009

Defense Date 06/24/2009

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag bisphosphonates,modified nucleotides,OAI-PMH Harvest,phosphorus

Language English

Advisor Prakash, G.K. Surya (committee chair), Olah, George A. (committee member), Rasul, Golam (committee member), Shing, Katherine S. (committee member)

Creator Email reha.ismail@gmail.com,rismail@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2413

Unique identifier UC1146775

Identifier etd-Ismail-3150 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-564078 (legacy record id),usctheses-m2413 (legacy record id)

Legacy Identifier etd-Ismail-3150.pdf

Dmrecord 564078

Document Type Dissertation

Rights Ismail, Rehana

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email uscdl@usc.edu

Abstract (if available)

Abstract This dissertation explores the field of organophosphorus chemistry. Phosphorus plays a major role in medicinal and natural product chemistry, which is the inspiration for the majority of the work presented in this dissertation.